InteractiveFly: GeneBrief

Nkx6/HGTX: Biological Overview | Regulation | Developmental Biology | Effects of Mutation and Overexpression | Evolutionary Homologs | References

Gene name - HGTX

Synonyms - Nkx6

Cytological map position - 70E3

Function - transcription factor

Keywords - CNS, motor neuron subtype, axon guidance

Symbol - HGTX

FlyBase ID: FBgn0040318

Genetic map position - 3L

Classification - Homeobox domain, NK decapeptide domain

Cellular location - nuclear

NCBI links: Entrez Gene

HGTX orthologs: Biolitmine

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).

A novel temporal identity window generates alternating Eve(+)/Nkx6(+) motor neuron subtypes in a single progenitor lineage

Spatial patterning specifies neural progenitor identity, with further diversity generated by temporal patterning within individual progenitor lineages. In vertebrates, these mechanisms generate 'cardinal classes' of neurons that share a transcription factor identity and common morphology. In Drosophila, two cardinal classes are Even-skipped (Eve)(+) motor neurons projecting to dorsal longitudinal muscles, and Nkx6(+) motor neurons projecting to ventral oblique muscles. Cross-repressive interactions prevent stable double-positive motor neurons. The Drosophila neuroblast 7-1 (NB7-1) lineage uses a temporal transcription factor cascade to generate five distinct Eve(+) motor neurons; the origin and development of Nkx6(+) motor neurons remains unclear. This study used a neuroblast specific Gal4 line, sparse labelling and molecular markers to identify an Nkx6(+) VO motor neuron produced by the NB7-1 lineage. Lineage analysis to birth-date the VO motor neuron to the Kr(+) Pdm(+) neuroblast temporal identity window. Gain- and loss-of-function strategies to test the role of Kr(+) Pdm(+) temporal identity and the Nkx6 transcription factor in specifying VO neuron identity. Lineage analysis identifies an Nkx6(+) neuron born from the Kr(+) Pdm(+) temporal identity window in the NB7-1 lineage, resulting in alternation of cardinal motor neuron subtypes within this lineage (Eve>Nkx6>Eve). Co-overexpression of Kr/Pdm generates ectopic VO motor neurons within the NB7-1 lineage - the first evidence that this TTF combination specifies neuronal identity. Moreover, the Kr/Pdm combination promotes Nkx6 expression, which itself is necessary and sufficient for motor neuron targeting to ventral oblique muscles, thereby revealing a molecular specification pathway from temporal patterning to cardinal transcription factor expression to motor neuron target selection. Thus this study shows that one neuroblast lineage generates interleaved cardinal motor neurons fates; that the Kr/Pdm TTFs form a novel temporal identity window that promotes expression of Nkx6; and that the Kr/Pdm > Nkx6 pathway is necessary and sufficient to promote VO motor neuron targeting to the correct ventral muscle group (Seroka, 2020).

Neural diversity from flies to mice arises from two major developmental mechanisms. First, neural progenitors acquire a unique and heritable spatial identity based on their position along the rostrocaudal or dorsoventral body axes. Second, temporal patterning based on neuronal birth-order results in individual progenitors producing a diverse array of neurons and glia. Temporal patterning is best characterized in Drosophila; neural progenitors (neuroblasts) located in the ventral nerve cord, central brain, and optic lobes all undergo temporal patterning, in which the neuroblast sequentially expresses a cascade of TTFs that specify distinct neuronal identities. Although all neuroblasts undergo temporal patterning, the TTFs are different in each region of the brain. Similar mechanisms are used in the mammalian cortex, retina, and spinal cord, although many TTFs remain to be identified (Seroka, 2020).

A major open question is how transient expression of TTFs like Kr and Pdm lead to long-lasting specification of molecular and morphological neuronal diversity. Good candidates for integrating spatial and temporal cues to consolidate motor neuron identity are homeodomain transcription factors expressed in post-mitotic motor neurons. In vertebrates, dorsoventral domains of the spinal cord are partitioned into 12 distinct cardinal classes of neurons - each characterized by development from a common progenitor domain, expression of unique homeodomain transcription factors with cross-repressive interactions to stabilize boundaries, and generating neurons with common morphology. This nomenclature is adapted to define Eve+ and Nkx6+ (Flybase: HGTX) motor neurons as two 'cardinal classes' of motor neurons: each class expresses a homeodomain transcription factor (Eve or Nkx6) with cross-repressive interactions, and each class consists of motor neurons with related neuronal morphology (Eve+ motor neurons project to dorsal and lateral longitudinal muscles; Nkx6+ motor neurons project to ventral muscle groups) (Seroka, 2020).

The Drosophila neuroblast 7-1 (NB7-1) is arguably the best characterized system for understanding TTF expression and function. Similar to most other ventral nerve cord neuroblasts, NB7-1 expresses the canonical TTF cascade Hb-Kr-Pdm-Cas with each TTF inherited by the GMCs born during an expression window, and transiently maintained in the two post-mitotic neurons produced by each GMC. The TTF cascade generates diversity among the five Eve+ U1-U5 motor neuron progeny of NB7-1: Hb specifies U1 and U2, Kr specifies U3, Pdm specifies U4, and Pdm/Cas together specify U5. Identifying TTF target genes, including transcription factors and cell surface molecules, will provide a comprehensive view of how developmental determinants direct neuronal morphology and synaptic partner choices (Seroka, 2020).

It has long been thought that the cardinal classes of motor neurons derive from distinct progenitors; Eve+ motor neurons derive from NB7-1, NB1-1, and NB4-2 whereas Hb9+ or Nkx6+ motor neurons derive from NB3-1 and others. However, DiI labeling of NB7-1 identified a potentially unknown motor neuron innervating ventral muscles, which is distinct from dorsal and lateral longitudinal muscles targeted by the Eve+ motor neurons. The observed ventral projection in this lineage could reflect transient exuberant outgrowths that are lost during larval life, or they could be due to an uncharacterized motor neuron that forms stable synapses with ventral muscles (Seroka, 2020).

This study shows that a newly discovered Kr/Pdm TTF window generates an Nkx6+ Eve-motor neuron, born between U3 and U4 in the NB7-1 lineage, that projects to ventral oblique (VO) muscles. It was also shown that overexpression of Kr/Pdm together, or Nkx6 alone, generates ectopic VO motor neurons based on molecular marker expression. Finally, this study demonstrates that Nkx6 is required for proper motor neuron axon targeting to ventral oblique muscles. These results establish a genetic pathway from TTFs (Kr/Pdm), to a cardinal motor neuron transcription factor (Nkx6) to motor axonal targeting. Also the unexpected discovery was made that a single progenitor can alternate production of different cardinal motor neuron classes (Seroka, 2020).

Kr/Pdm co-expression has been detected in several neuroblast lineages, but until now there has not been evidence that this TTF combination could specify neuronal identity. Previous work showed that the Kr/Pdm window generates a Kr/Pdm GMC, and this study shows that this GMC generates an Nkx6+ ventral-projecting motor neuron. It is unknown whether Kr/Pdm directly or indirectly activate nkx6 expression. The nkx6 gene lies in a 45kb region devoid of genes, and there are only a few, sparse predicted Kr or Pdm binding sites in this genomic expanse. How do Kr and Pdm together specify one fate (Nkx6+ VO motor neuron) whereas Kr or Pdm alone specify completely different fates (Eve+ dorsal motor neurons)? It is likely that Kr/Pdm together activate a different suite of target genes than either alone. For example, Kr/Pdm together may directly activate nkx6 expression, whereas neither alone has that potential. The emergence of single cell transcriptome and ChIP studies will help to reveal how the combination of Kr/Pdm TTFs generates different cell fate output compared to Kr or Pdm alone (Seroka, 2020).

The production of an Nkx6+ VO motor neuron in Kr/Pdm window interrupts the sequential production of Eve+ dorsal motor neurons in the NB7-1 lineage, resulting in an Eve > Nkx6 > Eve alternation of cardinal motor neuron production within the lineage. This is unusual, as in most cases neurons with similar morphology or function are produced together in a lineage. In mammals, progenitors generate neurons first, followed by glia; no examples are known of neuron>glia>neuron production from a single lineage. Similarly, Drosophila central brain neuroblast lineages produce the mushroom body γ neurons, then α'/ β' neurons, and lastly α/β neurons, with no evidence for alternating or interspersed fates. In the abdominal NB3-3 lineage, the early-born cells are in a mechanosensitive circuit, whereas the late-born cells are in a proprioceptive circuit. The only possible example of interleaved production of two morphological classes of neurons is in the Drosophila lateral antennal lobe neuroblast lineage, which alternate between uniglomerular and multiglomerular (AMMC) projection neurons. The use of clonal and temporal labeling tools will be needed to examine additional lineages to determine the prevalence of lineages producing temporally interleaved neuronal subtypes as in the NB7-1 lineage (Seroka, 2020).

Overexpression of Kr/Pdm or Nkx6 can induce only 2-3 ectopic VO motor neurons within the NB7-1 lineage. Clearly not all neurons in the lineage are competent to respond to these transcription factors. Early-born temporal identities specified by Hb and Kr (U1-U3) are unaffected by Kr/Pdm or Nkx6 overexpression, which is similar to previous data showing that early temporal fates are not affected by overexpression of later TTFs in multiple lineages. It remains a puzzle why the Kr+ U3 neuron does not switch to a VO fate upon overexpression of Kr/Pdm. There may need to be an equal level of Kr and Pdm to specify VO fate, although this would not explain why Kr/Pdm overexpression converts the Pdm+ U4 motor neuron to a VO fate. Alternatively, there may be an early chromatin landscape that blocks access to relevant Pdm target loci (Seroka, 2020).

It is noted that the assay of VO neuronal identity was done in newly-hatched larvae. Although motor circuits are functional at this time, larvae grow for five more days. These is no data on whether the ectopic VO motor neurons are functional or are maintained through the life of the larvae. This would be an important question for the future (Seroka, 2020).

Nkx6 and Eve have cross-repressive interactions (Broihier, 2004), but with some limitations: early-born Eve+ motor neurons are not affected by Nkx6 overexpression. Wild type animals even show sporadic expression of Nkx6 in the Eve+ U2 motor neuron, but in these neurons it has no effect on Eve expression, nor does it promote targeting to ventral oblique muscles. There appears to be a mechanism to block endogenous or overexpressed Nkx6 function in the early lineage of neuroblasts producing Eve+ motor neurons. The mechanism 'protecting' early-born Eve+ neurons from Nkx6 repression of Eve is unknown. Early lineages may lack an Nkx6 cofactor; Nkx6 could act indirectly via an intermediate transcription factor missing in early lineages; the early TTFs Hb or Kr may block Nkx6 function; or the eve locus could be in a subnuclear domain inaccessible to Nkx6 (Seroka, 2020).

Nkx6 promotes motor neuron specification in both Drosophila and vertebrates. In Drosophila, loss of Nkx6 reduces ventral projecting motor neuron numbers and increases the number of Eve+ neurons, while overexpression increases ventral projecting motor neuron numbers at the expense of Eve+ neurons. In vertebrates the Nkx6 family members Nkx6.1/Nkx6.2 appear to play a broader role in motor neuron specification. Nkx6.1/Nkx6.2 show early expression throughout the pMN domain; mice mutant for both Nkx6 family members lack most somatic motor neurons; and Nkx6.1 overexpression in chick or zebrafish can induce ectopic motor neurons. It would be interesting to investigate whether vertebrate Nkx6.1/Nkx6.2 are required to suppress a specific motor neuron identity, similar to the antagonistic relationship between Nkx6 and Eve in Drosophila (Seroka, 2020).

Neuroblasts in all regions of the Drosophila CNS (brain, ventral nerve cord, optic lobe) use TTF cascades to generate neuronal diversity, yet less is known about TTF target genes. It is likely that TTFs induce expression of suites of transcription factors that persist in neurons and confer their identity. Examples may include the 'morphology transcription factors' that specify adult leg motor neuron dendrite projections, but in this case it remains unknown whether these transcription factors control all other aspects of adult motor neuron identity. It is possible that 'morphology transcription factors' are one module downstream of a broader regulatory tier similar to the terminal selector genes in C. elegans (Seroka, 2020).

This study has identified a linear pathway from Kr/Pdm to Nkx6 which specifies VO motor neuron identity. TTFs could act by two non-mutually exclusive mechanisms: inducing a stable combinatorial codes of transcription factors that consolidate neuronal identity, or by altering the chromatin landscape to have a heritable, long lasting effect on motor neuron gene expression. The observation that Nkx6 is maintained in the VO neuron after fading of Kr/Pdm expression supports the former mechanism. Identification of Kr/Pdm or Nkx6 target genes would give a more comprehensive understanding of TTF specification of neuronal identity (Seroka, 2020).

The results presented in this work lead to several interesting directions. Other embryonic VNC lineages exhibit a Kr/Pdm window; does this window generate neurons in these lineages? Are there common features to neurons born in the Kr/Pdm window? Furthermore, do ectopic VO neurons make functional presynapses with the ventral oblique muscles, and do they have the normal inputs to their dendritic postsynapses? In only a few cases has it been shown the TTF-induced neurons are functionally integrated into the appropriate circuits [30]. Kr and Pdm orthologs have been identified in vertebrates. Looking for dual expression of Kr and Pdm orthologs in vertebrates may reveal a role in specifying temporal identity, similar to evidence for Hb and Cas TTFs having vertebrate orthologs that specify temporal identity (Seroka, 2020).


Transcriptional Regulation

Nk(x)-type homeobox genes are an evolutionarily conserved family that regulate diverse developmental processes. A novel Drosophila gene, Nk6, is described which encodes an Nk-type transcription factor most homologous to vertebrate Nkx6.1 and Nkx6.2. The homeodomains and NK decapeptide domains of all three proteins are highly conserved. Nk6 is expressed in the embryonic brain, ventral nerve cord, hindgut, and internal head structures. Nerve cord expression is in midline precursors, several ventral and intermediate column neuroblasts, and later in neurons but not glia, similar to the known expression of Nkx6 genes in the neural tube. Nk6 is positively regulated, directly or indirectly, by vnd in brain precursors. In vnd mutants, head neuroectoderm Nk6 expression is abolished where it is normally co-expressed with vnd. Conversely, vnd-overexpression leads to ectopic Nk6 expression in the brain. These findings further highlight the importance of interactions between Nk(x)-type genes in regulating their expression (Uhler, 2002).

The complexity of Nk6 expression in the CNS likely reflects the activities of multiple regulators. Candidates for Nk6 regulation include the early CNS patterning genes, vnd and sim, which co-localize with Nk6 mRNA in the head and midline respectively shortly after sim and vnd are activated. It was asked whether either of these genes regulate Nk6 by monitoring the distribution of Nk6 transcripts in embryos where sim and vnd expression is perturbed. sim transcripts are expressed in midline precursors from the cellular blastoderm (stage 5) onwards, and is required for proper midline cellular and molecular differentiation. Nk6 expression is abolished specifically in the midline of sim mutant embryos, suggesting that sim positively regulates Nk6, directly or indirectly, while Nk6 expression in the adjacent neuroblast layer is maintained (Uhler, 2002).

Mutant and misexpression assays were used to assess whether vnd regulates Nk6 expression. In the head, Nk6 is activated within an hour of Vnd protein expression: Nk6 is activated during gastrulation (stage 6), while Vnd is expressed from stage 5 onwards. In wild-type embryos Nk6 expression in the head localizes within the Vnd expression domain, with the later exception of a single neuroblast per lobe which begins expressing Nk6 at stage 10. Nk6 expression is affected in vnd mutants as early as stage 6, where expression in the head neuroectoderm is not activated. At later embryonic stages, all brain expression is absent apart from in the isolated Vnd-negative neuroblasts and their progeny. Anterior neuroectodermal expression in the nerve cord is never initiated. Nk6 expression in nerve cord neuroblasts is reduced and disordered in vnd mutants, with reductions typically occurring at ventral positions where neuroblasts normally co-express Nk6 and Vnd. The significance of this effect is uncertain, since many ventral neuroblasts in the nerve cord do not form and residual neuroblasts often switch to intermediate identities in vnd mutants (Uhler, 2002).

Whether vnd can alter Nk6 expression was examined by misexpressing vnd at different times during development. vnd was ubiquitously expressed at early stages, using heat-shock overexpression beginning during gastrulation. As early as stage 8, Nk6 mRNA expression in the head neuroectoderm is slightly expanded compared to wild-type, while expression in the nerve cord neuroectoderm is unchanged. Next, vnd was misexpressed throughout the neuroblast layer using the Gal4-UAS system. The sca-Gal4 driver directs transgene expression between stages 9 and 13. The number of cells expressing Nk6 increases in the brains of sca-Gal4 x UAS-vnd embryos by stage 11. While ectopic vnd is detected throughout the CNS of these embryos, ectopic Nk6 expression is restricted to only a subset of Vnd-expressing cells, suggesting that not all cells are competent to express Nk6 (Uhler, 2002).

Drosophila Nk6 has several general features in common with its two vertebrate counterparts. Expression of all three Nk(x)6 family members in the embryonic CNS is restricted to neurons. In Drosophila, chick, and mouse embryos, Nk(x)6 genes are transiently expressed in the ventral CNS midline early during development. During early stage 10, Drosophila Nk6 is expressed in most ventral column neuroblasts, similar to early mouse and chick Nkx6.1 and Nkx6.2 expression in the ventral third of the neural tube (p3, pMN and p2 domains). In Drosophila, a small subset of intermediate neuroblasts also express Nk6, paralleling the expression of chick and mouse Nkx62 expression in a narrow stripe of intermediate progenitors (p1). An important divergence between fly and vertebrate expression patterns is that broad CNS expression of the fly homolog begins relatively late during neurogenesis. In chick and mouse embryos, Nkx6.1 and Nkx6.2 are among the earliest genes expressed. Expression is initiated as longitudinal columns in the neural plate. The fly neuroectoderm, the equivalent of the neural plate, expresses Nk6 only in very anterior regions, with earliest expression occurring at stage 6 (Uhler, 2002).

Despite extensive double labeling analyses, the lineages and movements of cells that express Nk6 are not known. Although four neuroblasts in the ventral nerve cord which express the gene strongly between stages 10 and 11 of embryonic development have been identified, the identity of Nk6-positive cells thereafter have not been identified. It is likely that transcripts are expressed in some, though not all, GMC progeny of Nk6- positive neuroblasts, because several Nk6-positive GMCs and neuroblasts are positioned in a manner typical of GMCs budding off the parent neuroblast. Conversely, in at least one lineage, NB 4-2->GMC4-2a->RP2, Nk6 expression is not expressed beyond the neuroblast. The rapid expansion of Nk6 expression between stages 12 and 13 suggests that Nk6 expression is not lineage restricted (Uhler, 2002).

Sim and Vnd are expressed in the right cells and at appropriate times to potentially regulate Nk6 expression in distinct CNS domains. The absence of midline transcripts in sim mutants suggests that Nk6 is positively regulated by sim, as are most genes expressed in the midline. Vertebrate sim homologs are not expressed in the floorplate but in cells flanking the floorplate, several days after Nkx6.1 and Nkx6.2 activation, suggesting some evolutionary divergence in Nk(x)6 gene regulation (Uhler, 2002).

In the head, vnd likely positively regulates Nk6 expression. Both gene products co-localize within an hour of detectable Vnd. Nk6 expression in the head neuroectoderm and progeny is abolished in the absence of vnd, with the exception of two isolated neuroblasts that do not express Vnd. Conversely, overexpression of vnd leads to increased Nk6 expression in the head. Although effects on Nk6 expression in the ventral nerve cord are observed, the significance of the results are uninterpretable (Uhler, 2002).

There is a surprising degree of conservation in the dorso-ventral expression pattern of the fly Nk6 gene and its vertebrate homologs in the neural tube. However, the genes that regulate their expression in the CNS may be quite divergent, as there is no evidence that sim or Nkx2.2 are upstream of Nkx6 genes in the vertebrate CNS. Nkx6 expression is activated ventrally by Sonic hedgehog, repressed dorsally by BMP-7, and regulated across the antero-posterior axis by unknown notochord factors. Evidence in the mouse neural tube suggests that Nkx6.1 represses Nkx6.2 in the ventral region after an initial overlap. In the pancreas, however, analysis of single and double Nkx2.2/Nkx6.1 mutants indicates that Nkx2.2 is upstream of Nkx6.1, suggesting that the regulatory interaction between Nk(x)2 and Nk(x)6 type genes is evolutionarily conserved. Mouse Nkx2.2 can bind to the Nkx6.1 enhancer in vitro (Uhler, 2002).

Although the deduced amino acid sequence of Nk6, its expression pattern, and similarity to its vertebrate homologs suggest a role in regulating cell fates, the function of Nk6 has not been elucidated yet. Functional studies have been initiated using two independent deficiencies that cover the Nk6 locus. In trans-heterozygous Nk6-deficient embryos, axon scaffold defects, most commonly incomplete separation of the anterior and posterior commissures, have been found. This effect is a hallmark of abnormal midline glial development and is consistent with Nk6 expression in midline precursors (Uhler, 2002).

To test whether Hh might regulate Nkx6 expression in the fly, Nkx6 expression was examined in Hh mutants: they lack neuroblasts in rows 2, 5 and 6, including Nkx6-positive neuroblast 2-2. The Nkx6 expression pattern in the remaining five Nkx6-positive neuroblasts was wild type, suggesting that flies utilize a different mechanism from vertebrates to establish Nkx6 expression. Whether Hh was required for formation of motoneurons derived from an Nkx6-positive neuroblast was tested by examining co-expression of HB9 and phosphorlyated MAD (pMAD; Marques, 2002) in the RP1,3,4,5 motoneuron progeny of neuroblast 3-1. No change was found in these motoneurons in homozygous hh mutant embryos, suggesting that Hh signaling is unnecessary for their formation (Cheesman, 2004).

Targets of Activity

During early CNS development, Nkx6 is co-expressed with Ventral nervous system defective (Vnd) in a subset of medial column NBs, prompting an investigation of the genetic relationship between vnd and Nkx6. Vnd expression marks medial column CNS NBs and is required for the development of these cells. Nkx6 and Vnd expression were compared in wild-type embryos. Surprisingly, while Nkx6 and Vnd are co-expressed in a subset of medial column NBs, their expression patterns are otherwise complementary. At stage 9, Nkx6 is expressed in CNS midline precursors, while Vnd is expressed in ventral neuroectoderm flanking the midline. During stage 10, low-level Nkx6 expression initiates in five Vnd-positive NBs per hemisegment. At stage 11, Vnd and Nkx6 are expressed in non-overlapping groups of GMCs and postmitotic neurons. Notably, at this stage clusters of Nkx6-expressing cells are nestled within stripes of Vnd-expressing cells. The complementary patterns of Nkx6 and Vnd in GMCs and neurons are maintained throughout embryogenesis. These data raised the possibility that opposing activities of Nkx6 and vnd help establish and maintain their respective expression patterns (Broihier, 2004),

To investigate whether the complementary expression patterns of Nkx6 and Vnd arise due to their opposing activities, it was asked if vnd misexpression represses Nkx6. These analyses focus on the genetic relationship between Nkx6 and vnd in postmitotic neurons since these genes exhibit mutually exclusive patterns in these cells. The elav-GAL4 driver was used to express vnd in postmitotic neurons and it was found that this abolishes CNS expression of Nkx6. It was not possible to obtain meaningful loss-of-function data for vnd because nearly all medial column NBs and their progeny, many of which are Nkx6-positive, fail to develop in vnd mutant embryos. The requirement of vnd to promote medial column NB formation inhibited the ability to assay the effect of removing vnd function on Nkx6. Nevertheless, the ability of vnd misexpression to abolish Nkx6 expression supports the model that vnd represses Nkx6 to help establish the complementary expression patterns of Nkx6 and Vnd (Broihier, 2004),

In the reciprocal experiment, it was found that postmitotic misexpression of Nkx6 dramatically reduces the number of Vnd-positive neurons. Normally, 10.0±1.3 neurons express Vnd per hemisegment whereas only 4.2±1.8 neurons express Vnd per hemisegment (n=53) in Nkx6 misexpression embryos. However, Vnd expression is wild type in Nkx6 mutant embryos. Thus, Nkx6 is sufficient but not necessary to repress vnd expression (Broihier, 2004),

These data suggest that while high levels of Nkx6 and Vnd are cross-repressive in postmitotic neurons, these factors function in concert with other regulators during normal development to limit each other's expression. Given the similar expression profiles of Nkx6 and Hb9 and their independent regulation it was asked whether Nkx6 and hb9 act in parallel to repress vnd expression. As observed for Nkx6, hb9 misexpression in postmitotic neurons significantly reduces the number of Vnd-positive CNS neurons while hb9 mutants exhibit wild-type Vnd expression. However, removal of both hb9 and Nkx6 leads to an overproduction of Vnd-positive neurons; 13.6±2.1 Vnd-positive neurons (n=41) develop in double mutant embryos relative to ten in wild type. These results show that hb9 and Nkx6 act in parallel to repress vnd, and support the model that the complementary patterns of Nkx6 and vnd arise at least in part due to their opposing activities (Broihier, 2004),

The regulatory relationship between Nkx6 and hb9 -- both of which are expressed in ventrally projecting motoneurons -- and the dorsal motoneuron determinant eve was explored. eve and hb9 engage in a cross-repressive relationship to maintain their expression in distinct neuronal populations (Broihier, 2002). Since Nkx6 and Eve are also expressed in non-overlapping populations of neurons, it was asked whether they repress each other. It was first asked whether eve is sufficient to repress Nkx6 by misexpressing eve in all postmitotic neurons. Eve misexpression results in a near complete suppression of Nkx6 expression by embryonic stage 16, demonstrating that eve is sufficient to repress Nkx6 (Broihier, 2004),

In a reciprocal manner, misexpression of Nkx6 in all postmitotic neurons severely reduces Eve expression in the U MNs and EL neurons, though Eve expression in RP2 and aCC/pCC appears grossly normal. However, as observed for vnd, Eve expression is normal in Nkx6 mutant embryos. Thus, Nkx6 is sufficient but not necessary to repress eve (Broihier, 2004),

Nkx6 and hb9 also act in parallel to repress eve. Stage 15 hb9 mutant embryos contain 19.4±2.0 Eve-positive neurons per hemisegment. This number represents an increase of two Eve-positive neurons relative to wild type (Broihier, 2002). Significantly, stage 15 hb9KK30 Nkx6D25 double mutant embryos display 24.0±3.7 Eve-positive neurons per hemisegment, representing an increase of six Eve-positive neurons relative to wild type. The ectopic Eve-positive neurons arise at multiple positions within the CNS, suggesting they develop from multiple NB lineages; however, a number are situated close to the midline. To confirm this phenotype is caused by loss of Nkx6 activity from an hb9 mutant background, double-stranded Nkx6 RNA was injected into hb9KK30 mutant embryos. An average of 24.8±5.9 Eve-positive neurons per hemisegment was found in these embryos, demonstrating that injection of Nkx6 RNA into hb9 mutants phenocopies the Eve phenotype observed in hb9KK30 Nkx6D25 mutants. The further increase of Eve-positive neurons in hb9 Nkx6 mutant embryos relative to hb9 mutant embryos demonstrates that Nkx6 and hb9 collaborate to repress Eve. hb9 and Nkx6 thus act together to limit the expression of eve, a key determinant of dorsally projecting MN identity (Broihier, 2004),

Protein Interactions

Consistent with the common role of Nkx6 family members in specifying motor neuron identity, this study shows that over-expression of Drosophila Nkx6 results in an increase in the number of Fasiclin II expressing motor neurons in the intersegmental nerve B branch. Dissection of the regulatory domains of Nkx6 using chimeric cell culture assays revealed the presence of two repression domains and a single activation domain within this transcription factor. As well as its conserved homeodomain, Nkx6 also has a candidate Engrailed homology 1 (Eh1) domain that is conserved among all NKx6 family members, through which vertebrate NKx6-type proteins bind the co-repressor Groucho. Paralleling previous reports that the Eh1 domain of Vnd and Ind are ineffective in Gal4 chimeric assays, this study found that the Eh1 domain of Nkx6 did not significantly enhance repression in Gal4 chimeric assays. However, when co-immunoprecipitation analyses was performed, it was found that Nkx6 can bind Groucho and that binding of Nkx6 to this co-repressor is modulated intra-molecularly. Full length Nkx6 interacted with Groucho poorly, because sequences at the carboxyl terminal of NKx6 interfere with Groucho binding, despite the presence of the Eh1 domain. In contrast, a carboxyl terminal Nkx6 deletion bound Groucho strongly. In keeping with the presence of an activation domain within Nkx6, it is also reported that Nkx6 can activate reporter expression driven by an Nkx6.1 enhancer that mediates auto-activation in transient transfection assays. The presence of multiple repression domains in Nkx6 supports Nkx6's role as a repressor, potentially using both Groucho-dependent and independent mechanisms. Thus, Nkx6 likely functions as a dual regulator in embryos (Syu, 2009).



The expression pattern of Nk6 mRNA was determined by in situ hybridization to whole-mount embryos. Expression is detected in the developing hindgut, ventral maxillary epidermis-derived head structures, and the developing CNS. Nk6 expression initiates at stage 6 as two bilateral clusters in the head neuroectoderm. By stage 8, expression is seen in the hindgut primordium and ventral midline precursors. Expression in the nerve cord neuroectoderm, which is limited to anterior segments, begins at stage 9. Ventral nerve cord neuroblasts begin expressing transcripts at stage 10. During early stage 10, transient weak expression occurs in most neuroblasts immediately flanking the midline (ventral neuroblasts) and some intermediate neuroblasts. Expression then becomes restricted to two bilateral clusters of 3¯5 neuroblasts per neuromere by the end of stage 10, concomitant with declining midline expression. Transcripts are detected in GMCs from late stage 10 onwards. By late stage 11, only one to two neuroblasts per hemineuromere express Nk6. Strong expression in the ventral maxillary epidermis can be distinguished from anterior neuroectodermal expression at this stage. Expression in the nerve cord shifts from a tightly clustered distribution to a disperse pattern during early stage 12. By stage 13 there are approximately 15¯20 Nk6-positive cells per hemineuromere. Expression in the CNS, head structures, and hindgut persists at least until late stage 16 (Uhler, 2002).

To identify which precursor cells express Nk6, embryos were double-labeled for Nk6 mRNA and cell-specific markers. First focus was placed on dorso-ventral CNS patterning genes. During early neurogenesis, the transcription factors Single-minded (Sim), Vnd, Intermediate neuroblasts defective (Ind), and Muscle specific homeobox (Msh) subdivide the CNS into midline, ventral, intermediate and lateral columns respectively. Nk6 is clearly expressed in midline precursors since it is co-expressed with Sim. During stage 10, Nk6 expression changes from weak paramedian expression to strong clustered expression, with approximately one to two cells per cluster co-expressing Nk6 mRNA and Vnd protein, and two cells per cluster co-expressing Nk6 and Ind. No Nk6 transcripts are detected lateral to the Ind column (Uhler, 2002).

Double-labeled embryos for Nk6 and three transcription factors, Engrailed, Achaete and Castor, were examined. Double labeling for Nk6 and Engrailed, which is expressed in the posterior of each neuromere, positioned the Nk6 -positive neuroblast clusters to the anterior half of each neuromere. Double-labeling for Nk6 and Achaete, expressed in MP2 and X neuroblasts at stage 10, revealed that Nk6 is expressed in a neuroblast directly anterior to MP2, either NB 3-1 or 2-2. One, occasionally two, Nk6-positive neuroblasts are located just lateral to MP2, NBs 3-2 and 4-2. Castor is expressed in seven neuroblasts per hemineuromere at stage 10 and 18 by stage 11. Nk6 and Castor do not co-localize during stage 10 (except at midline). By stage 11, they co-localize in NB 3-2, positioned just anterior to NB4-2 which expresses Nk6 but not Castor. Very weak co-expression is also detected in NB2-2 and 3-1. Together, these results suggest that Nk6 stage 10¯11 positive neuroblast clusters locate anteriorly in ventral and intermediate column neuroblasts, including NBs 2-2, 3-1, 3-2 and 4-2 (Uhler, 2002).

Since Nkx6.2 is expressed in adult glia, whether Drosophila Nk6 is expressed in embryonic glia as well as in neurons was assessed by double labeling for the specific neuroectodermal glial marker Repo. The midline glia, which develop from the mesectoderm, do not express Repo. Since Nk6 and Repo are not co-expressed at any embryonic stage, Nk6 expression is specific to neuronal cells (Uhler, 2002).

Next, embryos were double-labeled for Nk6 transcripts and several well-characterized markers of interneurons and motorneurons, Even-skipped (Eve), 22C10 (Futsch), and Fasciclin II (FasII). Sensory neurons, which are in the peripheral nervous system, do not express Nk6. At stage 11, the transcription factor Eve is expressed in the NB 4-2->GMC4-2a->RP2 lineage. Although Nk6 is expressed in the NB 4-2 parent, no transcripts are detected in any Eve-positive cells at this or later embryonic stages. Several pioneer neurons begin expressing 22C10 and/or FasII at stages 11 and 12 (including aCC, pCC, MP1, SP1, vMP2 and dMP2), none of which co-express Nk6. Due to poor resolution of in situ staining, it is possible that cells may co-express Nk6 and 22C10 or FasII at later stages (Uhler, 2002).

The position of Nk6-expressing neurons was determined using an antibody, BP102, which recognizes all CNS axons. At stage 16, Nk6 is expressed across the entire medio-lateral axis of the nerve cord. The majority of Nk6-positive cells are positioned at and below (ventral to) the level of the neuropile (Uhler, 2002).

To identify the neuronal cell types most likely to be affected by loss of Nkx6 function, Nkx6-specific antibodies were generated. Antibody specificity was demonstrated on embryos bearing deletions of the Nkx6 locus. Nkx6 exhibits a highly dynamic expression pattern within the embryonic CNS. Nkx6 is first expressed in CNS midline precursors at embryonic stage 9; this expression is transient and is extinguished by stage 10. At stage 10, relatively weak Nkx6 expression was detected in neuroblasts. To identify the Nkx6-positive NBs, Nkx6 expression was assayed relative to Svp-LacZ and Deadpan – characterized markers of NB identity. These experiments illustrate that NBs 1-1, 1-2, 2-2, 3-1, 3-2, 4-2 and 5-2 express Nkx6 at low to moderate levels. By stage 11, Nkx6 is expressed in medial clusters of approximately 15 cells. Based on their position and size, these cells appear to be a mixture of GMCs and postmitotic neurons. Beginning at stage 12, neurons in the intermediate and lateral regions of the CNS activate Nkx6 expression. By stage 14, Nkx6 is expressed in a complex pattern of 30-40 neurons in each hemisegment. Notably, Nkx6 expression levels vary dramatically and reproducibly between neurons in late-stage embryos. The dynamic pattern of Nkx6 expression in the CNS suggests Nkx6 may function in the development of specific CNS cell types (Broihier, 2004),

To establish the identity of Nkx6-positive neurons, Nkx6 expression was compared to markers of defined neuronal subsets. Whether Nkx6 is expressed in MN and interneuron populations was investigated. Nkx6 expression was compared to that of Odd-skipped (Odd); Nkx6 and Odd are co-expressed in the MP1 and dMP2 interneurons. It was then asked whether Nkx6 is present in distinct MN groups. To this end, Nkx6 expression was compared to that of Hb9 and Eve. Hb9 is expressed in ventrally and laterally projecting MNs while Eve is expressed in dorsally projecting MNs (Broihier, 2002; Landgraf, 1999; Odden, 2002). Like Hb9 and Eve, Nkx6 and Eve are also expressed in complementary patterns. In contrast, the majority of Nkx6-expressing cells express Hb9, although Nkx6 is expressed in slightly more neurons than Hb9. The extensive co-expression of Nkx6 and Hb9 suggests that Nkx6 is also expressed in ventrally projecting MNs. Confirming this, it was found that Nkx6 is co-expressed with a Lim3-taumyc transgene, a marker of RP 1,3,4,5 (RP MNs) -- a group of well-characterized ventrally projecting MNs. This analysis established that Nkx6 is expressed in both interneurons and ventrally projecting MNs (Broihier, 2004),

The co-expression of Nkx6 and hb9 in ventrally projecting MNs raised the possibility that they act in a linear genetic pathway to control the development of these MNs. In addition, vertebrate Nkx6.1 is expressed in MN progenitors and is necessary for the activation of Hb9 in postmitotic MNs (Sander, 2000a; Vallstedt, 2001). However, Nkx6 and Hb9 were found to be expressed independently of each other in the Drosophila CNS. Thus, if Nkx6 regulates neuronal fate, it does so independently of regulating hb9 transcription. Instead, the independent regulation of Nkx6 and hb9 combined with their similar expression profiles suggests they may act in parallel to regulate neuronal fate (Broihier, 2004),

Zebrafish and fly Nkx6 proteins have similar CNS expression patterns and regulate motoneuron formation

Drosophila Nkx6 expression is first detected during early neurogenesis (stage 9) in the nerve cord midline, and it is weakly expressed in ventral column neurectoderm of rostral segments. An hour later (early stage 10), Nkx6 midline and neurectoderm expression is downregulated. Nkx6 expression is restricted to six ventral column neuroblasts, rostrally located in each hemisegment. Consistent with Uhler (2002) Drosophila Nkx6 expression is detected in neuroblasts 2-2, 3-1, 3-2 and 4-2. Nkx6 expression was also detected in neuroblasts 1-1 and 1-2. By early stage 11, Nkx6 was downregulated in neuroblasts and expressed in ganglion mother cells (GMCs) and postmitotic neurons. From stage 14 to the end of gastrulation, Nkx6 is expressed in a segmentally reiterated pattern of CNS neurons. At stage 14, many of these Nkx6-positive cells also expressed the postmitotic motoneuron marker, pMAD suggesting that many, perhaps all, motoneurons are initially Nkx6-positive. However, at later stages Eve-positive motoneurons no longer expressed Nkx6, consistent with the observation that the CNS contains Nkx6-negative, pMAD-positive motoneurons. These results reveal that some Nkx6-positive motoneurons are derived from Nkx6-positive neuroblasts, and raise the possibility that other Nkx6-positive motoneurons are derived from Nkx6-negative neuroblasts. Therefore, it is likely that Nkx6 expression is differentially regulated in neuroblasts and motoneurons. These results also suggest that Eve-positive fly motoneurons are similar to fish PMNs in that they both downregulate Nkx6 expression during development (Cheesman, 2004).

Whether zebrafish or fly Nkx6 is sufficient to generate ectopic motoneurons was tested in fly embryos. Fly lines were created carrying UAS-nkx6.1 (zebrafish) or UAS-Nkx6 (fly) transgenes and sca-Gal4 was used to drive Nkx6 expression in neurectoderm and all neuroblasts. Endogenous Nkx6 is extinguished from neuroblasts by stage 12; in contrast, in embryos expressing sca-Gal4 and either the fly or the zebrafish transgene, Nkx6 expression is maintained at least through stage 13. Supernumerary motoneurons were assayed by molecular markers and motor projections. The segmental nerve B (SNb) motor nerve to ventral muscles was substantially thicker than in wild-type embryos, consistent with production of supernumerary motoneurons (Cheesman, 2004).

Embryos misexpressing fly or zebrafish Nkx6 genes were examined for changes in several motoneuron markers: Eve, which labels all dorsally projecting motoneurons, Islet and HB9, which label ventrally projecting motoneurons, and pMAD, a pan-motoneuron marker. Misexpression of either the fly or zebrafish gene resulted in supernumerary motoneurons and loss of interneurons in the fly CNS. Most supernumerary motoneurons were in the lateral cluster of HB9-positive, Islet-positive motoneurons. There was also occasional duplication of the Eve-positive RP2 motoneuron. It is concluded that Nkx6 is sufficient for formation of both ventrally projecting and dorsally projecting motoneurons. However, the phenotype of these embryos is complex. Some motoneurons appeared unaffected, for example the HB9-positive, Islet-positive RP1,3,4,5 motoneurons and one type was slightly decreased, the Eve-positive U motoneurons. Consistent loss of identified interneurons was seen, including the Eve-positive ELs and Islet-positive EWs. Interestingly, in transgenic animals, cells in the EW position often expressed pMAD, a definitive motoneuron marker, consistent with a transformation of these interneurons into motoneurons (Cheesman, 2004).

Supernumerary motoneurons might arise from neuroblast duplication or change within a neuroblast lineage. To test whether there were duplicated neuroblasts, sca>Nkx6 or sca>nkx6.1 fly embryos were examined using various markers including Engrailed, Odd-skipped, Vnd, and Ind. Normal numbers of neuroblasts were seen in both backgrounds, ruling out neuroblast duplication, and suggesting that ectopic motoneurons result from an alteration in neuroblast lineage, for example, an interneuron to motoneuron transformation or a switch in GMC identity (Cheesman, 2004).

To examine potential lineage effects, fly Nkx6 was expressed under the control of Eagle (Eg), which is expressed in neuroblast 7-3 and its progeny, the HB9-positive EW interneurons and GW motoneuron. The same number of Eg-positive, HB9-positive cells was seen in controls and embryos overexpressing Nkx6. However, more than twice as many of these cells expressed pMAD in embryos overexpressing Nkx6 than in controls, revealing that at least in the case of neuroblast 7-3 the supernumerary motoneurons arise within the lineage, presumably by changing EW interneurons into motoneurons (Cheesman, 2004).

Whether Nkx6 was necessary for fly motoneuron formation was tested by RNAi. No change was seen in the numbers of HB9 or pMAD-positive cells in embryos lacking Nkx6, suggesting that it is not required for motoneuron formation, consistent with the phenotype of Nkx6 mutants (Broihier, 2004; Cheesman, 2004).


To initiate a functional analysis of Nkx6, a P element insertion, P{JG[LacZ]} was identified inserted 4 KB downstream of the Nkx6 locus. P{JG[LacZ]} is an enhancer trap and a mutant allele of Nkx6; Nkx6 protein levels are greatly reduced in P{JG[LacZ]} homozygotes. Via imprecise excision of P{JG[LacZ]}, a 25 kB deletion was generated that removes the 3' end of the Nkx6 locus (Nkx6D25). Nkx6D25 homozygous embryos do not express Nkx6 RNA or protein, indicating that Nkx6D25 is a null allele of the Nkx6 locus. The deletion also removes CG13479, a predicted gene with a single 83 amino acid ORF situated 14 KB downstream of Nkx6. The CNS phenotypes observed in Nkx6D25 mutant embryos are attributed to the Nkx6 locus for four reasons. (1) CG13479 is unlikely to be an embryonically-expressed transcript since CG13479 expression is not detected in wild-type embryos via RNA in situ hybridization and the Berkeley Drosophila Genome Project has not identified any embryonic ESTs for CG13479. (2) The axonal phenotypes in Nkx6 mutant embryos are largely rescued by Nkx6 expression in the CNS. (3) It was possible to phenocopy the Eve phenotype that was observe in hb9KK30 Nkx6D25 double mutant embryos by injection of ds Nkx6 RNA into hb9 mutant embryos. (4) The cell fate and axonal outgrowth phenotypes observed in Nkx6D25 embryos are reciprocal to those observed when Nkx6 is misexpressed via the GAL4/UAS system (Broihier, 2004).

Whether hb9 and Nkx6 coordinate the specification of ventrally projecting MN identity was investigated. RP1,3,4,5 MNs are large Nkx6-positive cells that lie close to the midline and project their axons contralaterally to ventral muscles within ISNb. Since both Nkx6 and Hb9 are expressed in RP1,3,4,5 (Broihier, 2002), it was asked whether these neurons develop properly in hb9 Nkx6 double mutant embryos. Islet and Lim3 are markers of RP1,3,4,5 identity (Thor, 1997; Thor, 1999) and are expressed in MNs in embryos singly mutant for Nkx6 or hb9 (Broihier, 2002). However, expression of Islet and Lim3 in the RP1,3,4,5 MNs is strongly reduced in hb9 Nkx6 double mutant embryos. Interestingly, the requirement of Nkx6 and hb9 to promote Islet and Lim3 expression is relatively specific to these RP MNs, since Islet and Lim3 expression is otherwise grossly normal in these embryos. The absence of these early determinants of RP1,3,4,5 MN identity strongly suggests that RP MNs are specified incorrectly in the absence of Nkx6 and hb9 activity. Hence, Nkx6 and hb9 act in parallel to control the fate of distinct MN subsets. They collaborate to restrict the expression of Eve, a key determinant of dorsally projecting MN identity, and to promote the expression of Islet and Lim3 in a well-defined subset of ventrally projecting MNs. While these functions of Nkx6 and hb9 may be distinct, a model is favored that Nkx6 and hb9 promote ventrally projecting MN identity by repressing eve expression in RP MNs (Broihier, 2004),

Nkx6 is co-expressed with Hb9, Lim3, and Islet in populations of ventrally projecting MNs. Since hb9, lim3, and islet are known to be required for proper axon guidance of ventrally projecting axons (Broihier, 2002; Odden, 2002; Thor, 1997; Thor, 1999), it was asked whether Nkx6 is also necessary for the axonal development of this MN population. Using anti-Fas2 antibody to label motor axon pathways, it was found that two of the four major nerve branches that innervate ventral and lateral muscles exhibit highly penetrant phenotypes in Nkx6 mutant embryos. Specifically, both secondary branches of the ISN, ISNb and ISNd, are absent in a significant proportion of Nkx6 mutant hemisegments. Notably, the Nkx6-positive RP1,3,4,5 MNs project within ISNb. The ISNb phenotype in four allelic combinations of Nkx6 was quantified, including embryos transheterozygous for Nkx6D25 and an unrelated deficiency of the region. The ISNb was scored as completely absent when axon extension into the ventral muscle field could not be detected. Likewise, ISNb was scored as reduced if any axons grew into the ventral muscle field, even if they initially bypassed their normal choicepoint. In all allelic combinations, defects were found in ISNb outgrowth in at least half of all hemisegments. In fact, for dNkx6D25 homozygotes or Nkx6D25/Df(3L)fz-D21 transheterozygotes, the penetrance is greater than 90%. The penetrance of the ISNd phenotype is roughly equivalent to that of ISNb. Hence, Nkx6 activity promotes proper axonal development of ISNb and ISNd (Broihier, 2004),

To ensure that loss of Nkx6 activity is responsible for the observed axonal phenotypes, whether Nkx6 misexpression in a Nkx6 mutant background rescues the ISNb outgrowth phenotype was examined. Targeted transposition was used to engineer an Nkx6GAL4 enhancer trap from the Nkx6P[JG(LacZ)] enhancer trap. The Nkx6GAL4 driver was used to express Nkx6 in Nkx6GAL4/Nkx6D25 mutant embryos. Nkx6 expression was found to be sufficient to rescue ISNb outgrowth in 72% of hemisegments, compared to 27% in the absence of Nkx6 misexpression. The ability of Nkx6 expression to largely rescue the observed motor axon phenotypes provides strong evidence that loss of Nkx6 is responsible for the axonal phenotypes in Nkx6D25 mutant embryos (Broihier, 2004),

Since Nkx6 and hb9 act in parallel to regulate neuronal fat, it was of interest to see whether they also act in parallel to regulate axon growth. However, the motor axon phenotypes in hb9 Nkx6 mutant embryos are nearly identical to those in Nkx6 mutants. Therefore, while Nkx6 and hb9 collaborate to regulate multiple neuronal fates, Nkx6 plays a specific non-redundant role to promote axonogenesis (Broihier, 2004),

To examine the role of Nkx6 during axonogenesis in more detail, focus was placed on axon projections of Hb9-positive neurons. Since Hb9 and Nkx6 are normally expressed in largely overlapping neuronal subsets, this enriched for Nkx6-positive axons relative to Fas2, which labels all motor axons. In wild-type embryos, Hb9-positive axons project in ISNb and synapse with their appropriate targets. However, no Hb9-positive axons were detected in ISNb in Nkx6D25 homozygous mutant embryos. In fact, few Hb9-positive axons are observed in the periphery of Nkx6 mutant embryos, suggesting these motor axons may remain in the nerve cords of Nkx6 mutants. To test this, Hb9-positive axons were followed in the nerve cords of Nkx6 mutant embryos. In wild type, Hb9-positive interneurons extend axons in multiple longitudinal fascicles in the CNS. In contrast, in Nkx6 mutant embryos, very few Hb9-positive axons were observed projecting along longitudinal fascicles. Since all Hb9-positive neurons appear to be specified in Nkx6 mutants, the motor axon phenotypes observed in Nkx6 mutants do not represent motoneuron to interneuron transformations. Rather, these data argue that Nkx6 potentiates axon growth of Nkx6-expressing neurons (Broihier, 2004),

The impaired axon extension observed in Nkx6 mutant embryos argues that Nkx6 is a positive mediator of axon growth. To test this model, axon growth was analyzed in embryos that over-expressed Nkx6 in all postmitotic neurons. To ensure high levels of Nkx6 expression in neurons, embryos carrying elavGAL4 and two copies of UAS-Nkx6 were used and focus was placed on ISNb axon projection. The overall pattern and thickness of ventral motor axon projections (including ISNb) is normal in these embryos suggesting that postmitotic overexpression of Nkx6 does not result in widespread transformations of neurons to the ventrally projecting MN fate. In support of this, Hb9 expression is wild type in elavGAL4:2XUAS-Nkx6 embryos. However, in this background a significant proportion of ISNb axons exhibit phenotypes consistent with overgrowth. For example, at least one ISNb branch with a clearly expanded terminal arbor was observed in 28% of hemisegments compared to 6% in wild type. Two phenotypes were observed in Nkx6 overexpression embryos that were never observed in wild type. Namely, excessive axonal branching was observed in ISNb in 14% of hemisegments. In 4% of hemisegments, ISNb axons from adjacent segments extend across the segment boundary and fuse together. These data support the conclusion that Nkx6 overexpression in ISNb-projecting neurons leads to axonal overgrowth, probably via the upregulation of molecules that promote axon growth and regulate guidance. Furthermore, the reciprocal effects of loss of function and overexpression of Nkx6 on axon growth argue that Nkx6 activates genes that promote axonogenesis (Broihier, 2004),

The preceding analysis indicates that Nkx6 promotes axon outgrowth of a subset of MNs. To investigate this possibility in more detail, the well-characterized axon projections of the RP1,3,4,5 MNs were followed in wild type and Nkx6 mutant backgrounds. A lim3-taumyc transgene (Thor, 1999) was used to follow RP motor axon projections in wild-type and Nkx6 mutant embryos. In wild type, it was possible to detect RP motor axons exiting the CNS in 86% of hemisegments scored. In contrast, it was possible to trace motor axons leaving the CNS in only 39% of hemisegments of Nkx6 mutant embryos. In most mutant hemisegments, the motor axons appeared thinner than in wild type. Furthermore, in 61% of Nkx6 mutant hemisegments, RP motor axons remain within the CNS, compared to 14% of wild type. The morphology of these truncated axons is often aberrant, suggesting their outgrowth has stalled. For example, enlarged growth cones were frequently observed with a club-like appearance. Finally, in 10% of mutant hemisegments, RP motor axons make dramatic guidance errors, often turning back inappropriately and extending toward the midline. These data demonstrate that Nkx6 activity is critical for proper growth and guidance of the RP1,3,4,5 MNs. Furthermore, the axon phenotypes exhibited by these MNs probably reflect a general requirement of Nkx6 in promoting axonogenesis of Nkx6-expressing neurons (Broihier, 2004),

The Nkx6 axonal phenotypes strongly suggest that Nkx6 regulates, probably directly, molecules that control axonal outgrowth and guidance. Fasciclin III (Fas3), a cell adhesion molecule, is a possible target of Nkx6 action in MNs since it is expressed by the RP1,3,4,5 MNs and promotes target recognition by their motor axons. Notably, Fas3 expression is strongly reduced in the RP MNs in Nkx6 mutant embryos relative to wild type. Consistent with a specific role for Nkx6 in regulating Fas3 expression, more lateral neurons that express Fas3 but not Nkx6 exhibit wild-type Fas3 expression in Nkx6 mutants. Together, these data show that Nkx6 promotes proper RP motor axon growth, and indicate that Nkx6 controls RP motor axon growth by regulating the transcription of adhesion and guidance molecules -- one of which is Fas3 (Broihier, 2004),

Ems and Nkx6 are central regulators in dorsoventral patterning of the Drosophila brain

In central nervous system development, the identity of neuroblasts critically depends on the precise spatial patterning of the neuroectoderm in the dorsoventral (DV) axis. This study has uncovered novel gene regulatory network underlying DV patterning in the Drosophila brain; the cephalic gap gene empty spiracles (ems) and the Nk6 homeobox gene (Nkx6) encode key regulators. The regulatory network implicates novel interactions between these and the evolutionarily conserved homeobox genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind) and muscle segment homeobox (msh). Msh cross-repressively interacts with Nkx6 to sustain the boundary between dorsal and intermediate neuroectoderm in the tritocerebrum (TC) and deutocerebrum (DC), and Vnd positively regulates Nkx6 by suppressing Msh. Remarkably, Ems is required to activate Nkx6, ind and msh in the TC and DC, whereas later Nkx6 and Ind act together to repress ems in the intermediate DC. Furthermore, the initially overlapping expression of Ems and Vnd in the ventral/intermediate TC and DC resolves into complementary expression patterns due to cross-repressive interaction. These results indicate that the anteroposterior patterning gene ems controls the expression of DV genes, and vice versa. In addition, in contrast to regulation in the ventral nerve cord, cross-inhibition between homeodomain factors (between Ems and Vnd, and between Nkx6 and Msh) is essential for the establishment and maintenance of discrete DV gene expression domains in the Drosophila brain. This resembles the mutually repressive relationship between pairs of homeodomain proteins that pattern the vertebrate neural tube in the DV axis (Seibert, 2009).

This study shows that the evolutionarily conserved homeodomain protein Ems is an integral component of the gene regulatory network that governs DV patterning in the posterior brain neuromeres, the TC and DC. This novel function is surprising because ems has hitherto been exclusively connected with patterning functions along the AP axis. It has been proposed that the combined activities of the gap genes ems, buttonhead and orthodenticle (ocelliless - FlyBase) generate head segments and that ems mutants exhibit defects in the formation of the intercalary and antennal segment as well as in the corresponding TC and DC in accordance with the early pattern of ems expression. ems probably also has a homeotic function in specifying aspects of intercalary segment identity. This study provides evidence that another crucial function of Ems is its cross-repressive interaction with Vnd. Previously, it was shown that vnd expression is dynamic and exhibits specific differences in the TC and DC. This study demonstrates that Ems is involved in the regulation of brain-specific differences in vnd expression, and that Vnd acts to repress ems in complementary parts of the TC and DC. These interactions help to refine the pattern into mutually exclusive domains at the onset of neurogenesis, which is important as both genes provide positional information that subsequently specifies the identity of individual brain NBs. Depending on the context, Vnd/Nkx2 can act as a transcriptional activator or repressor, as determined by physical interaction with the co-repressor Groucho, which enhances repression. Interestingly, it was observed that Ems also regulates the expression of two Nkx genes in an opposing manner: it represses vnd/Nkx2 but is necessary to activate Nkx6. The repressor function of Ems most likely also depends on Groucho; Ems has been reported to bind Groucho in vitro (Seibert, 2009).

In ems mutants, defects in proneural gene expression (lethal of scute and achaete) are restricted to NE regions where ems is normally expressed during early neurogenesis, leading to the loss of a subset of NBs in the TC and DC. This contrasts with the phenotype of the late embryonic ems mutant brain, which exhibits a severe reduction, or entire elimination, of the TC and DC, suggesting that the proper development of a larger NE domain and/or fraction of NBs in the TC and DC must be affected. However, in ems mutants the organization of the early procephalic NE appears normal until stages 9/10 and apoptosis is not detected. A possible explanation for the subsequent complete loss of TC and DC is that in ems mutants, vnd becomes derepressed in the ventral/intermediate NE of both neuromeres, and expression of msh, ind and Nkx6 is not activated. It has been shown that ectopic vnd prevents the expression of many NB identity genes. Indeed, the expression of a number of molecular markers has been reported to be absent in the ems mutant brain. It is therefore conceivable that in the TC and DC of ems mutants, as a consequence of lacking ems and ectopic vnd (and the absence of proneural gene activation), some NBs do not form. Additionally, owing to mis-specification of the NE (where neural identity gene expression is absent or altered), the other NBs and their progeny might still form but degenerate at later stages (Seibert, 2009).

It has been largely unclear how expression of Nkx6 is regulated in the brain NE, although Vnd has been suggested to act as a positive regulator. At the blastodermal stage, coexpression of ems and vnd is only observed in the intermediate and ventral NE of the TC and DC, which might account for early Nkx6 expression being limited to the respective NE in the brain and absent from the trunk. The data indicate that Ems and Vnd together facilitate the activation of Nkx6. Ems expression closely prefigures the domain of Nkx6 expression in the TC and DC, and together with the fact that Nkx6 is completely abolished in ems mutants, this suggests that Ems might act as a direct activator to regulate the extension of the Nkx6 domain along the AP axis. Vnd indirectly regulates the enlargement of the Nkx6 domain along the DV axis by repressing the Nkx6-repressor Msh. That DV patterning in the brain NE integrates AP signals is additionally supported by the fact that Ems is also necessary for activation of ind and msh, indicating that ems is a key regulator in DV patterning of the TC and DC. Evidence is also provided for a negative-feedback control in the DV regulatory network, in which Ems is needed to activate its own later-stage repressors, Nkx6 and Ind. Together, these data suggest not only that Ems regulates the expression of all DV genes (activating Nkx6, ind, msh and repressing vnd), but also that DV factors (Nkx6, Ind and Vnd) control expression of ems, indicating that integration of DV and AP patterning signals takes place at different levels in the DV genetic network (Seibert, 2009).

Nkx6 has been identified as specifically involved in DV patterning of the TC and DC. In addition to later suppression of ems (in concert with Ind), a further pivotal function of Nkx6 is to maintain the suppression of msh in the intermediate/ventral TC and DC that was initiated by Vnd. Since in both neuromeres the expression of Nkx6 starts before and persists longer than that of ind, and because msh is ventrally derepressed in Nkx6 but not in ind mutants, this implies that Nkx6 (but not Ind) is the major msh suppressor necessary to prevent intermediate/ventral NE and the descending NBs from adopting dorsal fates. Consequently, Nkx6 indirectly regulates the proper specification of brain NB identity by suppressing msh (and ems). Further experiments are required to show whether Nkx6 is also more directly involved in the fate specification of NBs and progeny cells in the brain, as has been shown in the VNC, where Nkx6 promotes the fate of ventrally projecting, and represses the fate of dorsally projecting, motoneurons (Seibert, 2009).

Additionally, cross-inhibitory interactions were observed between Nkx6 and Msh. It is assumed that this mutually repressive regulation in the TC and DC is necessary to stabilize the boundary between dorsal and intermediate NE, and to ensure the regionalized expression of msh and Nkx6 over time. It is likely that Nkx6 and Msh/Msx interact with the co-repressor Groucho to repress each other at the transcriptional level. Interestingly, aspects of the genetic interactions between Nkx6 and Msh/Msx seem to be evolutionarily conserved, since Msx1, which is expressed in the vertebrate midbrain and functions as a crucial determinant in the specification of dopamine neurons, represses Nkx6.1 in ventral midbrain dopaminergic progenitors of mice (Seibert, 2009).

It had not been shown until now that domains of DV gene expression in the Drosophila brain become established through cross-repressive regulation, and it is possible that such genetic interactions are more common than previously thought (e.g. Ind and Msh act as mutual inhibitors). This suggests that in the fly brain, cross-inhibition between pairs of homeodomain transcription factors is fundamental for establishing and maintaining DV neuroectodermal and corresponding stem cell domains. By contrast, in the NE of the VNC, where DV patterning is much better understood, cross-repressive interactions of homeobox genes are largely omitted. There, DV patterning is proposed to be conducted by a strict ventral-dominant hierarchy according to which ventral genes repress more-dorsal genes. However, one exception to the rule seems to be the cross-inhibitory interaction between Vnd and Ind. Interestingly, in the developing vertebrate neural tube, cross-repressive interactions of homeodomain proteins are common and indeed crucial for the establishment of discrete DV progenitor domains. This bears a marked resemblance to the mutually antagonistic relationship between pairs of homeodomain proteins that dorsoventrally pattern the fly brain (Seibert, 2009).

A predominant feature of the brain-specific DV genetic network described in this study, and a general design feature of gene regulatory networks, is the extensive use of transcriptional repression to regulate target gene expression in spatial and temporal dimensions. All factors involved in the network operate as repressors (except Ems, which may also serve as an activator), via mutual repression (between Ems and Vmd, and between Nkx6 and Msh), a double-negative mechanism (Vnd represses Msh, which represses Nkx6), and a negative-feedback loop (Ems is needed to activate Nkx6 and Ind, which in turn repress Ems). The spatial and temporal complexity of the regulatory interactions that have been deciphered implies similar complexity in the underlying cis-regulatory control of these factors. For example, the domain of msh expression is regulated by the input of at least two transcriptional repressors acting in subsequent time windows (Vnd early and Nkx6 late), and the input of at least three repressors regulates the dynamics of ems expression (Vnd early, Ind and Nkx6 late). The brain-specific DV patterning network probably comprises further genes in addition to those that have been identified, and it is likely that interactions with other putative regulators (e.g. Dorsal, Egfr, Dpp) will complement the present model. Altogether, these data provide the basis for a systematic comparison of the genetic processes underlying DV patterning of the brain between different animal taxa at the level of gene regulatory networks (Seibert, 2009).

The genetic factors considered in this study in the developing fly brain are expressed in similar NE domains from early embryonic stages onwards in the anterior neural plate in vertebrates. Emx2, for example, is expressed in the laterodorsal region, and Nkx2 genes in the ventral region, of the early vertebrate forebrain. At the four-somite stage (~E8), these two domains exhibit a common border, similar to that observed in Drosophila after Ems and Vnd have, through cross-repression, regulated their mutually exclusive expression domains. Moreover, whereas Msx genes are mainly expressed in dorsal regions of the posterior forebrain, midbrain and hindbrain, expression of Nkx6 genes is reported in more lateroventral regions, overlapping ventrally with the expression of Nkx2 genes. However, even though these patterns of gene expression exhibit certain similarities between insects and vertebrates, it remains to be shown whether their genetic interactions are also conserved (Seibert, 2009).

The Homeodomain Transcription Factor Hb9 Controls Axon Guidance in Drosophila through the Regulation of Robo Receptors

Transcription factors establish neural diversity and wiring specificity; however, how they orchestrate changes in cell morphology remains poorly understood. The Drosophila Roundabout (Robo) receptors regulate connectivity in the CNS, but how their precise expression domains are established is unknown. This study shows that the homeodomain transcription factor Hb9 acts upstream of Robo2 and Robo3 to regulate axon guidance in the Drosophila embryo. In ventrally projecting motor neurons, hb9 is required for robo2 expression, and restoring Robo2 activity in hb9 mutants rescues motor axon defects. Hb9 requires its conserved repressor domain and functions in parallel with Nkx6 to regulate robo2. Moreover, hb9 can regulate the mediolateral position of axons through robo2 and robo3, and restoring robo3 expression in hb9 mutants rescues the lateral position defects of a subset of neurons. Altogether, these data identify Robo2 and Robo3 as key effectors of Hb9 in regulating nervous system development (Santiago, 2014).

Combinations of transcription factors specify the tremendous diversity of cell types in the nervous system. Many studies have identified requirements for transcription factors in regulating specific events in circuit formation as neurons migrate, form dendritic and axonal extensions, and select their final synaptic targets. In most cases, the downstream effectors through which transcription factors control changes in neuronal morphology and connectivity remain unknown, although several functional relationships have been demonstrated (Santiago, 2014).

Conserved homeodomain transcription factors regulate motor neuron development across phyla. Studies in vertebrates and invertebrates have shown that motor neurons that project to common target areas often express common sets of transcription factors, which act instructively to direct motor axon guidance. In mouse and chick, Nkx6.1/ Nkx6.2 and MNR2/Hb9 are required for the specification of spinal cord motor neurons, and for axon pathfinding and muscle targeting in specific motor nerves. In Drosophila, Nkx6 and Hb9 are expressed in embryonic motor neurons that project to ventral or lateral body wall muscles, and although they are not individually required for specification, they are essential for the pathfinding of ventrally projecting motor axons. Axons that project to dorsal muscles express the homeodomain transcription factor Even-skipped (Eve), which regulates guidance in part through the Netrin receptor Unc5. Eve exhibits cross-repressive interactions with hb9 and nkx6, which function in parallel to repress eve and promote islet and lim3. Hb9 and Nkx6 act as repressors to regulate transcription factors in the spinal cord; however, guidance receptors that act downstream of Hb9 and Nkx6 have not been characterized. Interestingly, in both flies and vertebrates, Hb9 and Nkx6 are also expressed in a subset of interneurons, and knockdown experiments in Drosophila have suggested a role for hb9 in regulating midline crossing (Santiago, 2014).

Roundabout (Robo) receptors regulate midline crossing and lateral position within the developing CNS of invertebrates and vertebrates. Two recent studies in mice have also identified a role for Robos in regulating motor axon guidance in specific motor neuron populations. The three Drosophila Robo receptors have diversified in their expression patterns and functions. Robo2 is initially expressed in many ipsilateral pioneers and also contributes to Slit-mediated repulsion. Subsequently, robo2 expression is more restricted, and it is required to specify the medio-lateral position of axons. Robo3 is expressed in a subset of CNS neurons and also regulates lateral position (Santiago, 2014).

Characterization of the expression domains of the Drosophila Robos revealed an intriguing pattern, in which Robo1 is expressed on axons throughout the width of the CNS, Robo3 is found on axons in intermediate and lateral zones, and Robo2 is enriched on the most lateral axons. These patterns are transcriptional in origin, as replacing any robo gene with the coding sequence of another Robo receptor results in a protein distribution that matches the endogenous expression of the replaced gene (Spitzweck, 2010). A phenotypic analysis of these gene-swap alleles revealed the importance of transcriptional regulation for the diversification of robo gene function (Spitzweck, 2010). Robo2 and robo3's roles in regulating lateral position are largely dependent on their expression patterns, although unique structures within the Robo2 receptor are also important for its function in lateral position (Evans, 2010; Spitzweck, 2010). In the peripheral nervous system, the Atonal transcription factor regulates robo3 in chordotonal sensory neurons, directing the position of their axon terminals. In the CNS, the transcription factors lola and midline contribute to the induction of robo1. However, how the expression patterns of robo2 and robo3 are established to direct axons to specific medio-lateral zones within the CNS remains unknown (Santiago, 2014).

This study identifies a functional relationship between Hb9 and the Robo2 and Robo3 receptors in multiple contexts. Hb9 acts through Robo2 to regulate motor axon guidance and can direct the medio-lateral position of axons in the nerve cord through its effects on robo2 and robo3. Furthermore, hb9 interacts genetically with nkx6 and requires its conserved repressor domain to regulate robo2. Together, these data establish a link between transcriptional regulators and cell surface guidance receptors, providing an example of how upstream factors act through specific guidance receptors to direct circuit formation (Santiago, 2014).

This study has demonstrated a functional relationship between Hb9 and the Robo2 and Robo3 receptors in multiple contexts in the Drosophila embryo. In the RP motor neurons, hb9 is required for robo2 expression, and genetic rescue experiments indicate that robo2 acts downstream of hb9. Hb9 requires its conserved repressor domain and acts in parallel with Nkx6 to regulate robo2 and motor axon guidance. Moreover, hb9 contributes to the endogenous expression patterns of robo2 and robo3 and the lateral position of a subset of axons in the CNS, and can redirect axons laterally when overexpressed via upregulation of robo2. Finally, restoring Robo3 rescues the medial shift of MP1 axons in hb9 mutants, indicating that hb9 acts through robo3 to regulate medio-lateral position in a defined subset of neurons (Santiago, 2014).

Hb9 and nkx6 are required for the expression of robo2 in motor neurons, and rescue experiments suggest that the loss of robo2 contributes to the phenotype of hb9 mutants. However, nkx6 mutants and hb9 mutants heterozygous for nkx6 have a stronger ISNb phenotype than robo2 mutants, implying the existence of additional downstream targets. One candidate is the cell adhesion molecule FasIII, which is normally expressed in the RP motor neurons and appears reduced in nkx6 mutant embryos. Identifying the constellation of effectors that function downstream of Hb9 and Nkx6 will be key to understanding how transcription factors expressed in specific neurons work together to drive the expression of the cell surface receptors that regulate axon guidance and target selection (Santiago, 2014).

Robo2's activity in motor axon guidance appears distinct from the previously described activities of the Drosophila Robo receptors. Although Robo1 can replace Robo2's repulsive activity at the midline (Spitzweck, 2010), Robo2's function in motor axon guidance is not shared by either Robo1 or Robo3. Moreover, Robo2's antirepulsive activity at the midline and its ability to shift axons laterally when overexpressed both map to Robo2's ectodomain, whereas this study has found that Robo2's activity in motor axon guidance maps to its cytodomain (Evans, 2010; Spitzweck, 2010). The signaling outputs of Robo2's cytodomain remain unknown, as it lacks the conserved motifs within Robo1 that engage downstream signaling partners. How does Robo2 function during motor axon guidance? In mice, Robo receptors are expressed in spinal motor neurons and prevent the defasciculation of a subset of motor axons (Jaworski, 2012). Does Drosophila Robo2 regulate motor axon fasciculation? The levels of adhesion between ISNb axons and other nerves must be precisely controlled during the different stages of motor axon growth and target selection, and several regulators of adhesion are required for ISNb guidance. Furthermore, whereas Slit can be detected on ventral muscles, it is not visibly enriched in a pattern that suggests directionality in guiding motor axons, making it difficult to envision how Robo2-mediated repulsive or attractive signaling might contribute to ISNb pathfinding. Future work will determine how Robo2's cytodomain mediates motor axon guidance, whether this activity is Slit dependent, and whether Robo2 signals attraction, repulsion, or modulates adhesion in Drosophila motor axons (Santiago, 2014).

Elegant gene-swap experiments revealed the importance of transcriptional regulation in establishing the different expression patterns and functions of the Drosophila Robo receptors (Spitzweck, 2010). By analyzing a previously uncharacterized subset of axon pathways, this study has uncovered a requirement for Hb9 in regulating lateral position in the CNS. Although Hb9 can act instructively to direct lateral position when overexpressed, its endogenous expression in a subset of medially projecting neurons suggests that its ability to shift axons laterally is context dependent. A complex picture emerges in which multiple factors act in different groups of neurons to regulate robo2 and robo3. In a subset of interneurons, hb9 is endogenously required for lateral position through the upregulation of robo3 and likely robo2. In other neurons, such as those that form the outer FasII tracts, the expression patterns of robo2 and robo3 rely on additional upstream factors. What might be the significance of a regulatory network in which multiple sets of transcription factors direct lateral position in different groups of neurons? One possibility is that hb9-expressing neurons may share specific functional properties, such as the expression of particular neurotransmitters or ion channels. Alternatively, hb9 may regulate other aspects of connectivity. Indeed, Robo receptors mediate dendritic targeting in the Drosophila CNS, raising the exciting possibility that hb9 regulates both axonal and dendritic guidance through its effects on guidance receptor expression (Santiago, 2014).

What is the mechanism by which Hb9 regulates the expression of robo2, robo3, and its other downstream effectors? This study has found that Hb9 requires its conserved putative repressor domain and acts in parallel with Nkx6 to regulate robo2 and motor axon guidance. It has previously been shown that hb9 and nkx6 function in parallel to regulate several transcription factors. Hb9, nkx6 double mutants show decreased expression of islet and lim3 and upregulation of eve and the Nkx2 ortholog vnd. Are Hb9 and Nkx6 regulating robo2 or robo3 through any of their previously identified targets? Hb9 and nkx6 single mutants show no change in islet, lim3, or vnd expression, arguing that hb9 and nkx6 do not act solely through these factors to regulate robo2 or robo3. Eve expression is unaffected in nkx6 mutants, and whereas it is ectopically expressed in two neurons per hemisegment in hb9 mutants, these do not correspond to RP3 or MP1, the identifiable cells in which changes can be detected in robo2 and robo3. Therefore, the data do not support the hypothesis that Hb9 and Nkx6 regulate robo2 or robo3 primarily through their previously identified targets islet, lim3, vnd, or eve (Santiago, 2014).

Gain-of-function experiments in vertebrates suggest that Hb9 and Nkx6 act as repressors to regulate gene expression in the spinal. The finding that Hb9's Eh domain is required for motor axon pathfinding and robo2 regulation suggests that Hb9 acts as a repressor in this context as well, most likely through a previously unidentified intermediate target. In contrast, the Eh domain is not required for Hb9's ability to regulate robo3 or lateral position in hb9GAL4+ neurons that project to intermediate zones of the CNS. The finding that Hb9;delta;Eh retains significant activity in rescuing lateral position and robo3 expression indicates that Hb9 may regulate robo2 and robo3 via distinct mechanisms, perhaps involving different transcriptional cofactors or intermediate targets. In support of this hypothesis, hb9 overexpression in the ap neurons can induce robo2, but not robo3. These data raise the intriguing possibility that Hb9's ability to regulate robo2 and robo3 via different mechanisms contributed to the diversification of their expression patterns in the CNS. Determining how Hb9 and Nkx6 regulate their effectors will be key to achieving a complete understanding of how these conserved transcription factors control changes in cell morphology and axon pathfinding during development. Of note, Hb9 mutant mice exhibit defects in a subset of motor nerves, including the phrenic and intercostal nerves, which are also affected in Robo mutants. It will be of great interest to determine if despite the vast divergence in the evolution of nervous system development between invertebrates and vertebrates, Hb9 or Nkx6 has retained a role for regulating Robo receptors across species (Santiago, 2014).


A C. elegans Nkx6 homolog

The development of the reproductive system in C. elegans is a well-established model system for patterning and organogenesis. Mutation in the cog-1 gene cause novel phenotypes in late patterning in vulval lineages, establishment of the vulva-uterine connection, development and function of the spermathecal-uterine junction, and the development of vas deferens-proctodeal connection in the male. cog-1 was positionally cloned and found to encodes a homeobox protein most similar to the mammalian GTX and Nkx6.1 proteins. Analysis of cog-1 transcripts revealed that cog-1 is likely a complex locus with two promoters. Two mutant alleles of cog-1 differentially affect alternative transcripts and cause different phenotypes, suggesting that the two forms of cog-1 have distinct functions in C. elegans (Palmer, 2002).

The molecular mechanisms of differential pattern formation along the left/right (L/R) axis in the nervous system are poorly understood. The nervous system of the nematode C. elegans displays several examples of L/R asymmetry, including the directional asymmetry displayed by the two ASE taste receptor neurons, ASE left (ASEL) and ASE right (ASER). Although bilaterally symmetric in regard to all known morphological criteria, these two neurons display distinct chemosensory capacities that correlate with the L/R asymmetric expression of three putative sensory receptor genes, gcy-5, expressed only in ASER, and gcy-6 and gcy-7, expressed only in ASEL. In order to understand the genetic basis of L/R asymmetry establishment, a screen was performed for mutants in which patterns of asymmetric gcy gene expression are disrupted; a cascade of several symmetrically and asymmetrically expressed transcription factors was identified; these factors are sequentially required to restrict gcy gene expression to either the left or right ASE cell. These factors include the zinc finger transcription factor che-1; the homeobox genes cog-1, ceh-36, and lim-6; and the transcriptional cofactors unc-37/Groucho and lin-49. Specific features of this regulatory hierarchy are sequentially acting repressive interactions and the finely balanced activity of antagonizing positive and negative regulatory factors. A key trigger for asymmetry is the L/R differential expression of the Nkx6-type COG-1 homeodomain protein. These studies have thus identified transcriptional mediators of a putative L/R-asymmetric signaling event and suggest that vertebrate homologs of these proteins may have similar functions in regulating vertebrate brain asymmetries (Chang, 2003).

How left/right functional asymmetry is layered on top of an anatomically symmetrical nervous system is poorly understood. In the nematode C. elegans, two morphologically bilateral taste receptor neurons, ASE left (ASEL) and ASE right (ASER), display a left/right asymmetrical expression pattern of putative chemoreceptor genes that correlates with a diversification of chemosensory specificities. A previously undefined microRNA termed lsy-6 controls this neuronal left/right asymmetry of chemosensory receptor expression. lsy-6 mutants that were retrieved from a genetic screen for defects in neuronal left/right asymmetry display a loss of the ASEL-specific chemoreceptor expression profile with a concomitant gain of the ASER-specific profile. A lsy-6 reporter gene construct is expressed in less than ten neurons including ASEL, but not ASER. lsy-6 exerts its effects on ASEL through repression of cog-1, an Nkx-type homeobox gene, which contains a lsy-6 complementary site in its 3' untranslated region and that has been shown to control ASE-specific chemoreceptor expression profiles. lsy-6 is the first microRNA identified with a role in neuronal patterning, providing new insights into left/right axis formation (Johnston, 2003).

Molecular architecture of annelid nerve cord supports common origin of nervous system centralization in bilateria

To elucidate the evolutionary origin of nervous system centralization, the molecular architecture of the trunk nervous system was investigated in the annelid Platynereis dumerilii. Annelids belong to Bilateria, an evolutionary lineage of bilateral animals that also includes vertebrates and insects. Comparing nervous system development in annelids to that of other bilaterians could provide valuable information about the common ancestor of all Bilateria. The Platynereis neuroectoderm is subdivided into longitudinal progenitor domains by partially overlapping expression regions of nk and pax genes. These domains match corresponding domains in the vertebrate neural tube and give rise to conserved neural cell types. As in vertebrates, neural patterning genes are sensitive to Bmp signaling. These data indicate that this mediolateral architecture was present in the last common bilaterian ancestor and thus support a common origin of nervous system centralization in Bilateria (Denes, 2007).

Given the obvious paucity of information from the fossil record, the main strategy to elucidate CNS evolution is to compare nervous system development in extant forms. This comparative study of mediolateral neural patterning and neuron-type distribution in the developing trunk CNS of the annelid Platynereis revealed an unexpected degree of similarity to the mediolateral architecture of the developing vertebrate neural tube (Denes, 2007).

Three similarities are described. (1) The Platynereis and vertebrate neuroepithelium are similarly subdivided (from medial to lateral) into a sim+ midline and four longitudinal CNS progenitor domains (nk2.2+/nk6+, pax6+/nk6+, pax6+/pax3/7+, and msx+/pax3/7+), laterally bounded by an msx+, dlx+ territory. This strongly indicates a common evolutionary origin from an equally complex ancestral pattern. It is highly unlikely that precisely this mediolateral order and overlap in expression of orthologous genes in the CNS neuroectoderm should evolve twice independently. One can also discount the possibility that these genes are necessarily linked and thus co-opted as a package because they also act independently of each other in other developmental contexts (nk2.2 in endoderm development; pax6 in eye development, pax3/7 in segmentation, and msx in muscle development). Following similar reasoning, the complex conserved topography of gene expression along the anteroposterior axis in the enteropneust and vertebrate head is considered homologous (Denes, 2007).

(2) Evidence was found for conserved neuron types emerging from corresponding domains in Platynereis and in vertebrates. Serotonergic neurons involved in locomotor control form from the medial nk2.2+/nk6+ domain. A conserved population of hb9+ cholinergic somatic motoneurons emerges from the adjacent pax6+/nk6+ domain. Neurons expressing interneuron markers are found at the same level and more laterally, and single cells positive for sensory marker genes populate the lateral dlx+ domain. Notably, characterization of neuron types in the developing Platynereis nervous system is yet incomplete so that the full extent of conservation in neuron type distribution remains to be determined (Denes, 2007).

(3) Bmp signaling is similarly involved in the dose-dependent control of the neural genes. The finding that exogenous Bmp4 protein differentially regulates neural patterning genes in Platynereis nervous system development corroborates recent evidence that Bmps play an ancestral role in the mediolateral patterning of the bilaterian CNS neuroectoderm. Also, the strong upregulation of Pdu-atonal in the larval ectoderm goes in concert with Drosophila data that indicate that Dpp signaling positively regulates atonal expression in the lateral PNS anlage, and it supports the view that Bmp signaling also plays a conserved role in the specification of peripheral sensory neurons. Conservation of the molecular mediolateral CNS architecture concomitant with its sensitivity to Bmp signaling indicates that the developmental link between Bmp signaling and nervous system centralization predates Bilateria (Denes, 2007).

Taken together, these data make a very strong case that the complex molecular mediolateral architecture of the developing trunk CNS, as shared between Platynereis and vertebrates, was already present in their last common ancestor, Urbilateria. The concept of bilaterian nervous system centralization implies that neuron types concentrate on one side of the trunk, as is the case in vertebrates and many invertebrates including Platynereis, where they segregate and become spatially organized (as opposed to a diffuse nerve net). The data reveal that a large part of the spatial organization of the annelid and vertebrate CNS was already present in their last common ancestor, which implies that Urbilateria had already possessed a CNS (Denes, 2007).

Evolutionary conservation of the molecular mediolateral architecture as shared between Platynereis and vertebrates would imply that it was initially present also in the evolutionary lines leading to Drosophila, the nematode Caenorhabditis, and the enteropneust Saccoglossus. Yet it is clear from the available data that these animals are missing or have modified at least part of this pattern, although the extent of conservation may actually be larger than is currently apparent. For example, nk2.2/vnd and pax6 expression were costained in the fly, and a complementary pattern was found at germ-band-extended stage, reminiscent of the Platynereis and vertebrate situation. Strikingly, however, there is no trace so far of the conserved mediolateral architecture in the nematode Caenorhabditis and hardly any in the enteropneust Saccoglossus. How did this come about? Fly and nematode exhibit very fast development, making it plausible that they have (partially) omitted the transitory formation of longitudinal progenitor domains to speed up neurodevelopment. For the enteropneust, however, the situation is less clear. Why is the pattern absent in an animal that otherwise shows strong evolutionary conservation? One possible explanation is that the enteropneust trunk has lost part of its neuroarchitecture due to an evolutionary change in locomotion. While annelids and vertebrates propel themselves through trunk musculature (and associated trunk CNS), the enteropneust body is mainly drawn forward by means of the contraction of the longitudinal muscles in their anterior proboscis and collar. Possibly, enteropneusts have partially reduced their locomotor trunk musculature concomitant with motor parts of the CNS (while the peripheral sensory neurons prevailed in 'diffuse' arrangement). In line with this, expression of the conserved somatic motoneuron marker hb9/mnx is mostly absent from the Saccoglossus trunk ectoderm except for few patches. A more detailed understanding of enteropneust nervous system organization, neuron type distribution, and locomotion will help with resolving this issue (Denes, 2007).

An overall conservation of mediolateral CNS neuroarchitecture as proposed in this study does not imply that everything is similar. It is clear that the lines of evolution leading to annelids and vertebrates diverged for more than 600 million years, and numerous smaller or larger modifications of the ancestral pattern must have accumulated in both lines. The common-ground pattern as elucidated in this study helps in identifying these changes. For example, annelid and vertebrate differ in the deployment of gsx and dbx orthologs. While mouse gsh and dbx genes act early to specify interneuron progenitor domains in the dorsal neural tube, it was found the Platynereis gsx and dbx genes expressed at differentiation stages only. Adding to this, Pdu-gsx is expressed at a different mediolateral position in the nk2.2+ domain, and Pdu-dbx expression is much more restricted than that of its vertebrate counterparts (though the overall mediolateral coordinates correspond). It is hypothesized that these differences relate to the emergence of new interneuron domains (gsx+; dbx+) inside of the ancestral pax6+/pax3/7+ domain in the dorsal vertebrate neural tube. For this, it is conceivable that genes were recruited that had been active already in the differentiation of the diversifying interneuron populations. It is worth mentioning that the role of gsx in neuronal development also varies among vertebrates (Denes, 2007).

Homology of the vertebrate and Platynereis mediolateral molecular architecture is inevitably linked to the notion of dorsoventral axis inversion during early chordate evolution. In his 1875 essay on the origin of vertebrates Anton Dohrn discusses the resemblances between vertebrates and annelids and states that 'what stands most in the way of such a comparison has been the viewpoint that the nervous system of [annelids] is located in the venter, but that of vertebrates in the dorsum. Hence the one is called the ventral nerve cord, the other the dorsal nerve cord. Had we not possessed the terms dorsal and ventral, then the comparison would have been much easier.' How did the relocation of the trunk CNS from ventral to dorsal come about? Anton Dohrn proposed that vertebrate ancestors inverted their entire body dorsoventrally so that the former belly became the new back. This would not necessarily involve a sudden major shift in the lifestyle of an ancestor, as argued by critics of DV axis inversion. One can also imagine that an inversion involved transitional forms, with hemisessile or burrowing lifestyle and changing orientation toward the substrate. These animals had gill slits and lived as filter feeders. Only when early vertebrates left the substrate and acquired a free-swimming lifestyle would their new belly-up orientation have been fixed such that their CNS was then dorsal. Dohrn believed that the foremost gill slits then formed a new mouth on the new ventral body side. More than 130 years later, the molecular data on annelid neurodevelopment corroborate the key aspect of Dohrn's annelid theory, which is the homology of the annelid and vertebrate trunk CNS (Denes, 2007).

Isolation and charactherizationj of Nkx-6.1

The isolation, sequence and developmental expression in the central nervous system of several members of the chicken and mouse Nkx gene family is reported. These are among the earliest genes to be regionally expressed in the neural plate; they are expressed just above the axial mesendoderm (prechordal mesendoderm and notochord). Each Nkx gene has a distinct spatial pattern of expression along the anterior-posterior axis of the ventral central nervous system: Nkx-2. 2 is expressed along the entire axis, whereas Nkx-2.1 is restricted to the forebrain, and Nkx-6.1 and Nkx-6.2 are largely excluded from the forebrain. They are also expressed in distinct patterns along the dorsal-ventral axis. These genes are expressed in both the ventricular and mantle zones; in the mantle zone, Nkx-6.1 is co-expressed with Islet-1 in a subset of motor neurons. Like other Nkx genes, expression of Nkx-6.1 is induced by the axial mesendoderm and by sonic hedgehog protein. BMP-7 represses Nkx-6.1 expression. While the notochord can induce Nkx-6.1 expression in the anterior neural plate, sonic hedgehog protein does not, suggesting that the notochord produces additional molecules that can regulate ventral patterning (Qiu, 1998).

A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube

Distinct classes of neurons are generated at defined positions in the ventral neural tube in response to a gradient of Sonic Hedgehog (Shh) activity. A set of homeodomain transcription factors expressed by neural progenitors act as intermediaries in Shh-dependent neural patterning. These homeodomain factors fall into two classes: class I proteins are repressed by Shh and class II proteins require Shh signaling for their expression. The profile of class I and class II protein expression defines five progenitor domains, each of which generates a distinct class of postmitotic neurons. Cross-repressive interactions between class I and class II proteins appear to refine and maintain these progenitor domains. The combinatorial expression of three of these proteins (Nkx6.1, Nkx2.2, and Irx3) specifies the identity of three classes of neurons generated in the ventral third of the neural tube (Briscoe, 2000).

Ventral neural patterning by Nkx homeobox genes: Nkx6.1 controls somatic motor neuron fates and ventral interneuron fates

There is growing evidence that sonic hedgehog (Shh) signaling regulates ventral neuronal fate in the vertebrate central nervous system through Nkx-class homeodomain proteins. The patterns of neurogenesis in mice carrying a targeted mutation in Nkx6.1 have been examined. These mutants show a dorsal-to-ventral switch in the identity of progenitors and in the fate of postmitotic neurons. At many axial levels there is a complete block in the generation of V2 interneurons and motor neurons and a compensatory ventral expansion in the domain of generation of V1 neurons, demonstrating the essential functions of Nkx6.1 in regional patterning and neuronal fate determination (Sander, 2000a).

To define the role of Nkx6.1 in neural development, patterns of neurogenesis were compared in the embryonic spinal cord and hindbrain of wild-type mice and mice lacking Nkx6.1. In wild-type embryos, neural expression of Nkx6.1 is first detected at spinal cord and caudal hindbrain levels at about embryonic day 8.5 (E8.5; Qiu, 1998), and by E9.5 the gene is expressed throughout the ventral third of the neural tube. The expression of Nkx6.1 persists until at least E12.5. Nkx6.1 expression was also detected in mesodermal cells flanking the ventral spinal cord. To define more precisely the domain of expression of Nkx6.1, its expression was compared with that of 10 homeobox genes (Pax3, Pax7, Gsh1, Gsh2, Irx3, Pax6, Dbx1, Dbx1, Dbx2, and Nkx2.9>) that have been shown to define discrete progenitor cell domains along the dorsoventral axis of the ventral neural tube (Sander, 2000a).

This analysis revealed that the dorsal boundary of Nkx6.1 expression is positioned ventral to the boundaries of four genes expressed in dorsal progenitor cells: Pax3, Pax7, Gsh1, and Gsh2. Within the ventral neural tube, the dorsal boundary of Nkx6.1 expression is positioned ventral to the domain of Dbx1 expression and close to the ventral boundary of Dbx2 expression. The domain of Pax6 expression extends ventrally into the domain of Nkx6.1 expression, whereas the expression of Nkx2.2 and Nkx2.9 overlaps with the ventral-most domain of Nkx6.1 expression (Sander, 2000a).

To address the function of Nkx6.1 in neural development, progenitor cell identity and the pattern of neuronal differentiation were examined in Nkx6.1 null mutant mice. A striking change was detected in the profile of expression of three homeobox genes (Dbx2, Gsh1, and Gsh2) in Nkx6.1 mutants. The domains of expression of Dbx2, Gsh1, and Gsh2 each expand into the ventral neural tube. At E10.5, Dbx2 is expressed at high levels by progenitor cells adjacent to the floor plate, but at this stage ectopic Dbx2 expression is detected only at low levels in regions of the neural tube that generate motor neurons. By E12.5, however, the ectopic ventral expression of Dbx2 has become more uniform and now clearly includes the region of motor neuron and V2 neuron generation. Similarly, in Nkx6.1 mutants, both Gsh1 and Gsh2 are ectopically expressed in a ventral domain of the neural tube and also in adjacent paraxial mesodermal cells (Sander, 2000a).

The ventral limit of Pax6 expression is unaltered in Nkx6.1 mutants, although the most ventrally located cells within this progenitor domain express a higher level of Pax6 protein than those in wild-type embryos. No change was detected in the patterns of expression of Pax3, Pax7, Dbx1, Irx3, Nkx2.2, or Nkx2.9 in Nkx6.1 mutant embryos. Importantly, the level of Shh expression by floor plate cells is unaltered in Nkx6.1 mutants. Thus, the loss of Nkx6.1 function deregulates the patterns of expression of a selected subset of homeobox genes in ventral progenitor cells without an obvious effect on Shh levels. The role of Shh in excluding Dbx2 from the most ventral region of the neural tube appears therefore to be mediated through the induction of Nkx6.1 expression. Consistent with this view, ectopic expression of Nkx6.1 represses Dbx2 expression in chick neural tube (Briscoe, 2000). The detection of sites of ectopic Gsh1/2 expression in the paraxial mesoderm as well as the ventral neural tube, both sites of Nkx6.1 expression, suggests that Nkx6.1 has a general role in restricting Gsh1/2 expression. The signals that promote ventral Gsh1/2 expression in Nkx6.1 mutants remain unclear but could involve factors other than Shh that are secreted by the notochord (Sander, 2000a).

The domain of expression of Nkx6.1 within the ventral neural tube of wild-type embryos encompasses the progenitors of three main neuronal classes: V2, MN, and V3 interneurons. Whether the generation of any of these neuronal classes is impaired in Nkx6.1 mutants was examined, focusing first on the generation of motor neurons. In Nkx6.1 mutant embryos there is a marked reduction in the number of spinal motor neurons, as assessed by expression of the homeodomain proteins Lhx3, Isl1/2, and HB9 and by expression of the gene encoding the transmitter synthetic enzyme choline acetyltransferase. In addition, few if any axons were observed to emerge from the ventral spinal. The incidence of motor neuron loss, however, varied along the rostrocaudal axis of the spinal cord. Few if any motor neurons were detected at caudal cervical and upper thoracic levels of Nkx6.1 mutants analyzed at E11-E12.5, whereas motor neuron number was reduced only by 50%-75% at more caudal levels. At all axial levels, the initial reduction in motor neuron number persisted at both E12.5 and p0, indicating that the loss of Nkx6.1 activity does not simply delay motor neuron generation. Moreover, no increase was detected in the incidence of TUNEL+ cells in Nkx6.1 mutants, providing evidence that the depletion of motor neurons does not result solely from apoptotic death (Sander, 2000a).

The persistence of some spinal motor neurons in Nkx6.1 mutants raises the possibility that the generation of particular subclasses of motor neurons is selectively impaired. To address this issue, the expression of markers of distinct subtypes of motor neurons was monitored at both spinal and hindbrain levels of Nkx6.1 mutant embryos. At spinal levels, the extent of the reduction in the generation of motor neurons that populate the median (MMC) and lateral (LMC) motor columns was similar in Nkx6.1 mutants as assessed by the number of motor neurons that coexpressed Isl1/2 and Lhx3 (defining MMC neurons) and by the expression of Raldh2 (defining LMC neurons). In addition, the generation of autonomic visceral motor neurons was reduced to an extent similar to that of somatic motor neurons at thoracic levels of the spinal cord of E12.5 embryos. Thus, the loss of Nkx6.1 activity depletes the major subclasses of spinal motor neurons to a similar extent (Sander, 2000a).

At hindbrain levels, Nkx6.1 is expressed by the progenitors of both somatic and visceral motor neurons. Therefore whether the loss of Nkx6.1 might selectively affect subsets of cranial motor neurons was examined. A virtually complete loss in the generation of hypoglossal and abducens somatic motor neurons was detected in Nkx6.1 mutants, as assessed by the absence of dorsally generated HB9+ motor neurons. In contrast, there was no change in the initial generation of any of the cranial visceral motor neuron populations, assessed by coexpression of Isl1 and Phox2a within ventrally generated motor neurons. Moreover, at rostral cervical levels, the generation of spinal accessory motor neurons was also preserved in Nkx6.1 mutants. Thus, in the hindbrain the loss of Nkx6.1 activity selectively eliminates the generation of somatic motor neurons, while leaving visceral motor neurons intact. Cranial visceral motor neurons, unlike spinal visceral motor neurons, derive from progenitors that express the related Nkx genes Nkx2.2 and Nkx2.9. The preservation of cranial visceral motor neurons in Nkx6.1 mutant embryos may therefore reflect the dominant activities of Nkx2.2 and Nkx2.9 within these progenitor cells (Sander, 2000a).

Whether the generation of ventral interneurons is affected by the loss of Nkx6.1 activity was examined. V2 and V3 interneurons are defined, respectively, by expression of Chx10 and Sim1. A severe loss of Chx10 V2 neurons was detected in Nkx6.1 mutants at spinal cord levels, although at hindbrain levels of Nkx6.1, mutants ~50% of V2 neurons persist. In contrast, there is no change in the generation of Sim1 V3 interneurons at any axial level of Nkx6.1 mutants. Thus, the elimination of Nkx6.1 activity affects the generation of only one of the two major classes of ventral interneurons that derive from the Nkx6.1 progenitor cell domain (Sander, 2000a).

Evx1+, Pax2+ V1 interneurons derive from progenitor cells located dorsal to the Nkx6.1 progenitor domain within a domain that expresses Dbx2 but not Dbx1. Because Dbx2 expression undergoes a marked ventral expansion in Nkx6.1 mutants, whether there might be a corresponding expansion in the domain of generation of V1 neurons was examined. In Nkx6.1 mutants, the region that normally gives rise to V2 neurons and motor neurons now also generates V1 neurons, as assessed by the ventral shift in expression of the En1 and Pax2 homeodomain proteins. Consistent with this, there was a two- to three-fold increase in the total number of V1 neurons generated in Nkx6.1 mutants. In contrast, the domain of generation of Evx1/2 V0 neurons, which derive from the Dbx1 progenitor domain, was unchanged in Nkx6.1 mutants. Thus, the ventral expansion in Dbx2 expression is accompanied by a selective switch in interneuronal fates from V2 neurons to V1 neurons. In addition, some neurons within the ventral spinal cord of Nkx6.1 mutants coexpress the V1 marker En1 and the V2 marker Lhx3. The coexpression of these markers is rarely if ever observed in single neurons in wild-type embryos. Thus, within individual neurons in Nkx6.1 mutants, the ectopic program of V1 neurogenesis appears to be initiated in parallel with a residual, albeit transient, program of V2 neuron generation. This result complements observations in Hb9 mutant mice, in which the programs of V2 neuron and motor neuron generation coincide transiently within individual neurons (Sander, 2000a).

Taken together, these findings reveal an essential role for the Nkx6.1 homeobox gene in the specification of regional pattern and neuronal fate in the ventral half of the mammalian CNS. Within the broad ventral domain within which Nkx6.1 is expressed, its activity is required to promote MN and V2 interneuron generation and to restrict the generation of V1 interneurons. The idea is favored that the loss of MN and V2 neurons is a direct consequence of the loss of Nkx6.1 activity, since the depletion of these two neuronal subtypes is evident at stages when only low levels of Dbx2 are expressed ectopically in most regions of the ventral neural tube. Nonetheless, the possiblity that low levels of ectopic ventral Dbx2 expression could contribute to the block in motor neuron generation cannot be excluded. Consistent with this view, the ectopic expression of Nkx6.1 is able to induce both motor neurons and V2 neurons in chick neural tube. V3 interneurons and cranial visceral motor neurons derive from a set of Nkx6.1 progenitors that also express Nkx2.2 and Nkx2.9 (Briscoe, 1999). The generation of these two neuronal subtypes is unaffected by the loss of Nkx6.1 activity, suggesting that the actions of Nkx2.2 and Nkx2.9 dominate over that of Nkx6.1 within these progenitors. The persistence of some spinal motor neurons and V2 neurons in Nkx6.1 mutants could reflect the existence of a functional homolog within the caudal neural tube (Sander, 2000a).

The role of Nkx6.1 revealed in these studies suggests a model in which the spatially restricted expression of Nkx genes within the ventral neural tube has a pivotal role in defining the identity of ventral cell types induced in response to graded Shh signaling. Strikingly, in Drosophila, the Nkx gene NK2 has been shown to have an equivalent role in specifying neuronal fates in the ventral nerve cord. Moreover, the ability of Nkx6.1 to function as a repressor of the dorsally expressed Gsh1/2 homeobox genes parallels the ability of Drosophila NK2 to repress Ind, a Gsh1/2-like homeobox gene. Thus, the evolutionary origin of regional pattern along the dorsoventral axis of the central nervous system may predate the divergence of invertebrate and vertebrate organisms (Sander, 2000a).

Specification of neuronal fate in the vertebrate central nervous system depends on the profile of transcription factor expression by neural progenitor cells, but the precise roles of such factors in neurogenesis remain poorly characterized. Two closely related transcriptional repressors, Nkx6.2 and Nkx6.1, are expressed by progenitors in overlapping domains of the ventral spinal cord. Tenetic evidence that differences in the level of repressor activity of these homeodomain proteins underlies the diversification of interneuron subtypes, and provides a fail-safe mechanism during motor neuron generation. A reduction in Nkx6 activity further permits V0 neurons to be generated from progenitors that lack homeodomain proteins normally required for their generation, providing direct evidence for a model in which progenitor homeodomain proteins direct specific cell fates by actively suppressing the expression of transcription factors that direct alternative fates (Vallstedt, 2001).

Effect of Nkx6.1 and Nkx6.2 mutation

The Nkx homeobox genes are expressed in a variety of developing tissues and have been implicated in controlling tissue patterning and cell differentiation. Expression of Nkx6.2 (Gtx) was previously observed in the embryonic neural tube, testis, and differentiating oligodendrocytes. To investigate the role of Nkx6.2 in the control of cell specification and differentiation, mice with null mutations in Nkx6.2 were generated using the standard gene targeting approach. Null mutant mice are viable and fertile without apparent histological and immunohistochemical changes in the central nervous systems and testis. The absence of detectable phenotypes suggests a redundant function of Nkx6.2 in mouse development (Cai, 2001).

During early neural development, the Nkx6.1 homeodomain neural progenitor gene is specifically expressed in the ventral neural tube, and its activity is required for motoneuron generation in the spinal cord. Nkx6.1 also controls oligodendrocyte development in the developing spinal cord, possibly by regulating Olig gene expression in the ventral neuroepithelium. In Nkx6.1 mutant spinal cords, expression of Olig2 in the motoneuron progenitor domain is diminished, and the generation and differentiation of oligodendrocytes are significantly delayed and reduced. The regulation of Olig gene expression by Nkx6.1 is stage dependent; ectopic expression of Nkx6.1 in embryonic chicken spinal cord results in an induction of Olig2 expression at early stages, but an inhibition at later stages. Moreover, the regulation of Olig gene expression and oligodendrogenesis by Nkx6.1 also appears to be region specific. In the hindbrain, unlike in the spinal cord, Olig1 and Olig2 can be expressed both inside and outside the Nkx6.1-expressing domains and oligodendrogenesis in this region is not dependent on Nkx6.1 activity (Liu, 2003).

Although many studies have focused on the mechanisms of motoneuron specification, little is known about the factors that control the subsequent development of postmitotic motoneurons. The transcription factor Nkx6.1 is required for the early specification of somatic motoneuron progenitors in the spinal cord. The present analysis of hindbrain motoneuron development in Nkx6.1-deficient mouse embryos reveals that the early specification of branchio-motoneurons is independent of Nkx6.1 function, but that it is required for their subsequent development. In Nkx6.1 mutant mice, defects are observed in the migration, as well as in the axon projections of branchio-motoneurons. A detailed analysis of the migratory defect in facial branchio-motoneurons reveals ectopic expression of the cell surface receptors Ret and Unc5h3 in premigratory neurons, but no changes in the rhombomeric environment. Taken together, these findings demonstrate a requirement for Nkx6.1 in the development of postmitotic motoneurons, and suggest a cell-autonomous function in the control of branchio-motoneuron migration (Müller, 2003).

Groucho-mediated transcriptional repression establishes progenitor cell pattern and neuronal fate in the ventral neural tube

The pattern of neuronal specification in the ventral neural tube is controlled by homeodomain transcription factors expressed by neural progenitor cells, but no general logic has emerged to explain how these proteins determine neuronal fate. Most of these homeodomain proteins possess a conserved eh1 motif that mediates the recruitment of Gro/TLE corepressors. The eh1 motif underlies the function of these proteins as repressors during neural patterning in vivo. Inhibition of Gro/TLE-mediated repression in vivo results in a deregulation of cell pattern in the neural tube. These results imply that the pattern of neurogenesis in the neural tube is achieved through the spatially controlled repression of transcriptional repressors -- a derepression strategy of neuronal fate specification (Muhr, 2001).

Graded inductive signals specify cell fates in a position-dependent manner in the neural tube. Within the ventral neural tube, the identities of neural progenitor cells are assigned initially by the actions of Sonic hedgehog (Shh). Graded Shh signaling establishes distinct ventral progenitor domains by regulating the spatial pattern of expression of a set of homeodomain (HD) proteins that comprise members of the Pax, Nkx, Dbx, and Irx families. These HD proteins can be subdivided into class I and class II proteins based on their differential regulation by Shh signaling. The class I proteins are expressed by neural progenitor cells in the absence of Shh signaling, and their expression is repressed by Shh. In contrast, the expression of the class II proteins depends on exposure to Shh (Muhr, 2001 and references therein).

How do these HD proteins specify neuronal fate. The establishment of progenitor cell identity appears to involve cross-regulatory interactions between complementary pairs of class I and class II HD proteins that share a common boundary. These interactions define the spatial extent of individual progenitor domains and establish sharp boundaries between adjacent domains, thus ensuring that cells within individual domains express distinct combinations of HD proteins. The profile of class I and class II HD protein expression within a progenitor cell appears to direct neuronal fate. Most strikingly, several of these progenitor HD proteins have the ability to induce the ectopic generation of neuronal subtypes when misexpressed outside the confines of their normal progenitor domains. The inductive activities of these progenitor HD proteins involve the activation of expression of downstream transcription factors that serve intermediary roles in the determination of neuronal fate. In addition, gene targeting studies in mice have established the essential role of many of these class I and class II proteins in the specification of ventral neuronal identity (Muhr, 2001 and references therein).

Eight of the ten progenitor HD proteins implicated in ventral neural patterning share a motif related to the core eh1 region of the Engrailed repressor (EnR) domain. This motif mediates in vitro interactions of class I and class II HD proteins with Groucho-TLE (Gro/TLE) corepressors, and underlies the function of these proteins as repressors in neural patterning in vivo. Disruption of Gro/TLE function in neural cells in vivo leads to an impairment of ventral patterning. Three conclusions have been reached: (1) there is a common mechanism of action of the class I and class II progenitor HD proteins involved in ventral patterning; (2) Gro/TLE corepressors play a role in patterning the ventral neural tube; (3) the spatial pattern of neurogenesis in the ventral neural tube is achieved through the repression of repressors (Muhr, 2001).

To identify functional domains that mediate the neural patterning activity of the Nkx proteins, a focus was placed on a conserved ~10 amino acid motif, termed the TN, or NK decapeptide, domain. Nkx2.2, Nkx2.9, Nkx6.1, Nkx6.2 and Drosophila Ventral nervous system defective (Vnd) each possess a TN domain. This domain shows sequence similarity to the core region of the engrailed homology-1 (eh1) domain present in Engrailed (En), a transcriptional repressor. The eh1 motif interacts with Gro/TLE corepressors, and Gro/TLE proteins can bind to certain Nkx class proteins (Muhr, 2001).

The idea that Gro/TLE corepressors mediate the neural patterning activity of progenitor HD proteins currently rests on three lines of evidence: (1) the presence of an eh1 domain in class I and class II proteins underlies their Gro/TLE binding activity in vitro, and is required for their repressor functions in vivo; (2) Gro/TLE genes are expressed in the ventral neural tube at the time that neural pattern is established; (3) Grg5, a protein that inhibits Gro/TLE repressor function, deregulates the pattern of progenitor HD protein expression and blocks ectopic neuronal specification in vivo (Muhr, 2001).

The dorsal expansion in the domains of expression of the class II proteins Nkx6.1 and Nkx2.2 observed after Grg5 expression provides evidence that Gro/TLE function is required normally to establish the p1/p2 and pMN/p3 progenitor domain boundaries (MN refering to motor neuron). Expression of Grg5 also disrupted the normal mutual exclusion in the domains of expression of the class I/class II protein pairs Dbx2/Nkx6.1 and Pax6/Nkx2.2. Thus, a reduction in Gro/TLE activity blocks the ability of class II proteins to repress class I protein expression. However, there is not a ventral expansion in the domains of expression of the class I proteins Dbx2 and Pax6. One possible explanation for this asymmetry in HD protein deregulation is that a higher level of Gro/TLE activity is required for the repressor activity of the class I proteins than for the class II proteins. In addition, the detection of higher levels of ventral Gro/TLE gene expression than dorsal implies that expression of Grg5 will be more effective in reducing the net level of Gro/TLE activity in dorsal than ventral regions of the neural tube, favoring a dorsal expansions in progenitor HD protein expression. The early onset of Nkx6.1 expression, together with a higher level of ventral Gro/TLE gene expression, may also explain why Grg5 expression blocks the Nkx6.1-mediated induction of ectopic MNs, but is not able to inhibit the generation of MNs within the pMN domain. The findings with Grg5 support an essential role for Gro/TLE proteins in neural patterning, but there is still a need to define changes in neuronal fate that occur after elimination of the Gro/TLE proteins themselves (Muhr, 2001).

Pax6, in contrast to most other class I proteins, lacks an eh1 domain and functions as an activator. Nevertheless, Pax6 represses Nkx2.2 expression in vivo, implying that its function at the pMN-p3 boundary is achieved through an intermediary repressor. The finding that the domain of Nkx2.2 expression expands dorsally upon Grg5 overexpression implies that this intermediary repressor itself functions in a Gro/TLE-dependent manner. Taken together, these observations suggest that the establishment and maintenance of ventral progenitor domains -- whether achieved by direct repression or by activation of intermediary repressors -- depend on the activity of Gro/TLE corepressors (Muhr, 2001).

The finding that the activity of progenitor HD proteins depends on Gro/TLE-mediated repression provides several insights into the strategies used to establish neuronal diversity in the central nervous system. Focus is placed here on how transcriptional repression mediates the functions of the class II repressor proteins Nkx6.1 and Nkx2.2, although similar arguments apply for many of the class I proteins. The class II proteins Nkx6.1 and Nkx2.2 are required for the generation of MNs and V3 neurons, respectively. These activities appear to be achieved through the expression of downstream determinants of neuronal subtype identity. For example, within the pMN domain, Nkx6.1 promotes the expression of MNR2, a dedicated MN determinant. Nkx6.1 functions as a repressor during the specification of MNs in dorsal regions of the neural tube, favoring the idea that Nkx6.1 controls the expression of MNR2 within the pMN domain itself through its role as a repressor of class I proteins, although this remains to be established. In this view, the loss of MNs in Nkx6.1 mutant mice results from the ectopic ventral expression of class I proteins rather than from the loss of an Nkx6.1 activator function (Muhr, 2001).

How do Nkx6.1 and Nkx2.2 induce MNs and V3 neurons along the entire dorsoventral axis of the neural tube? In ventral progenitor cells, the inductive activities of Nkx6.1 and Nkx2.2 appear to depend on their ability to act as repressors of their complementary class I proteins, Dbx2 and Pax6. But in the dorsal neural tube, progenitor cells lack expression of many of the ventral class I repressor proteins. Thus, dorsal neural progenitors must also express repressors of MN and V3 neuronal differentiation -- repressors that are themselves subject to repression by Nkx6.1 or Nkx2.2. The identity of the dorsal repressors of MN and V3 neuron generation is not known, but the Gsh1/2 HD proteins are plausible candidates as suppressors of MN specification. Both Gsh proteins possess an eh1 motif (see Supplemental table) and are normally restricted to the dorsal neural tube, but are ectopically expressed ventrally in mouse Nkx6.1 mutants (Muhr, 2001).

The class II proteins also inhibit alternative neuronal fates within their normal domains of expression. Within the pMN and p2 domains, the expression of Nkx6.1 prevents V1 interneuron generation, and within the p3 domain, Nkx2.2 expression prevents MN generation. Thus, the Nkx proteins promote certain neuronal fates and block others, even though both activities are mediated primarily through repression. This reliance on repression distinguishes the function of Nkx proteins in neural fate specification from that of many other transcription factors whose roles in the selection of cell fates appears to reflect a combination of activator and repressor functions. The expression of Nkx2.2 and Nkx6.1 persists in certain post-mitotic neurons, and thus it remains possible that putative activator functions of these proteins are relevant for aspects of neuronal differentiation other than those examined in this study. Indeed, in other regions of the developing nervous system, the Phox2 HD proteins have been shown to function as activators of neuronal differentiation genes (Muhr, 2001).

How is neuronal fate decided when two repressor HD proteins are coexpressed within individual neural progenitor cells? Within the p3 domain, cells coexpress Nkx6.1 and Nkx2.2, yet the activity of Nkx2.2 is dominant, and progenitors generate V3 neurons rather than MNs. One conceivable reason for this is that Nkx2.2 has a higher affinity than Nkx6.1 for Gro/TLE proteins and thus sequesters available Gro/TLE corepressor activity, preventing Nkx6.1 function. Against this idea, Dbx2 is ectopically expressed in p3 domain progenitors in Nkx6.1 mutants, indicating that Nkx6.1 still functions as a repressor in this domain. A second and more plausible explanation is that Nkx2.2 blocks MN generation in p3 progenitors at a step downstream of progenitor HD proteins by repressing the expression of MN subtype determinants. Thus, instances of coexpression of class I and/or class II repressor proteins within progenitor cells may reflect the selection of neuronal fate through repression at the level of downstream neuronal subtype determinants rather than at the level of progenitor HD proteins (Muhr, 2001).

Taken together, these findings favor a model in which the pattern of neuronal specification is achieved primarily through the selectivity of repressor interactions with cis-acting DNA sequences present in the regulatory regions of different progenitor HD proteins and neuronal subtype determinants. This model requires that repressor HD proteins with distinct activities in neuronal specification recognize distinct DNA target sequences. In support of this idea, the class II proteins Nkx2.2 and Nkx6.1 have different patterning activities in the neural tube, possess divergent HDs, and recognize distinct target DNA sequences. In addition, the finding that hybrid class I and class II proteins consisting solely of the HD fused to the EnR or TN domain mimic the activity of the full-length proteins indicates that the distinct activities of class II and class I repressor proteins in neural patterning are likely to reside in the specificity of DNA recognition encoded in the HD (Muhr, 2001).

The finding that class II proteins and most class I proteins function as repressors leaves unresolved the issue of the role of transcription factors that activate the expression of neuronal subtype determinants. The results imply that progenitor cells arrayed along the entire dorsoventral axis of the neural tube possess a latent potential for activation of expression of all neuronal subtype determinants. In an extreme view, these subtype determinants may be activated by a single common activator protein that is expressed in a uniform manner along the entire dorsoventral axis of the neural tube. The ability of such an activator to induce different subtype determinant genes would then be constrained by the repertoire of cis-acting binding sites for class I and class II HD protein repressors present in their regulatory regions. This view argues that the specificity of neuronal subtype generation emerges largely from the patterned expression of repressors (Muhr, 2001).

In principle, it is possible to consider an alternative view in which distinct activator proteins are expressed within individual progenitor domains, with these activators operating upstream of but in a linear pathway with neuronal subtype determinants such as MNR2. In this view, the patterns of expression of these upstream activators would themselves need to be defined by the repressor activities of the class I and class II HD proteins. But the question of what activates the domain-restricted expression of these upstream activators immediately resurfaces. Thus, at its root, the activation of subtype determinants along the dorsoventral axis of the neural tube is likely to be a spatially unrestricted process. Clarification of this issue will require the identification of proteins that activate the expression of neuronal subtype determinants (Muhr, 2001).

In this context, it is intriguing that several basic helix-loop-helix (bHLH) transcriptional factors are expressed in discrete domains along the dorsoventral axis of the neural tube. Some of these genes transgress progenitor domain boundaries, whereas others are restricted to individual progenitor domains. Studies of bHLH protein function in vertebrates have begun to suggest that these proteins can influence neuronal subtype identity, in addition to their more general roles in neurogenesis. Determining whether and how the activity of bHLH proteins is integrated with progenitor HD protein-mediated repression during the specification of neuronal fate may help in the further dissection of mechanisms of ventral neuronal patterning (Muhr, 2001).

This analysis of the function of progenitor HD proteins has focused on neuronal specification along the dorsoventral axis of the neural tube. There are also clear restrictions in the potential for neuronal generation along the rostrocaudal axis of the neural tube. It is noteworthy that many HD proteins implicated in rostrocaudal neural patterning—including other Pax and Nkx proteins, and the Gsh, Msx, Gbx, and Tlx proteins—also possess eh1-like domains. Indeed, in a sample of 165 vertebrate HD proteins, many expressed by neural cells, ~36% were found to possess an eh1 domain (see Supplemental table). Gro/TLE-dependent repression may, therefore, have a more pervasive role in establishing precise spatial patterns of neuronal generation along both major axes of neural tube development. In addition, since homologs of the Nkx, Msx, and Gsh proteins control neuronal patterning along the dorsoventral axis of the Drosophila CNS, these results suggest that Gro/TLE-mediated corepression may be an evolutionarily conserved step in CNS patterning (Muhr, 2001).

Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain

The genetic program that underlies the generation of visceral motoneurons in the developing hindbrain remains poorly defined. The roles of Nkx6 and Nkx2 (Drosophila homolog: Vnd) class homeodomain proteins in this process were examined; evidence is provided that these proteins mediate complementary roles in the specification of visceral motoneuron fate. The expression of Nkx2.2 in hindbrain progenitor cells is sufficient to mediate the activation of Phox2b, a homeodomain protein required for the generation of hindbrain visceral motoneurons. The redundant activities of Nkx6.1 and Nkx6.2, in turn, are dispensable for visceral motoneuron generation but are necessary to prevent these cells from adopting a parallel program of interneuron differentiation. The expression of Nkx6.1 and Nkx6.2 is further maintained in differentiating visceral motoneurons, and consistent with this the migration and axonal projection properties of visceral motoneurons are impaired in mice lacking Nkx6.1 and/or Nkx6.2 function. This analysis provides insight also into the role of Nkx6 proteins in the generation of somatic motoneurons. Studies in the spinal cord have shown that Nkx6.1 and Nkx6.2 are required for the generation of somatic motoneurons, and that the loss of motoneurons at this level correlates with the extinguished expression of the motoneuron determinant Olig2. Unexpectedly, it has been found that the initial expression of Olig2 is left intact in the caudal hindbrain of Nkx6.1/Nkx6.2 compound mutants, and despite this, all somatic motoneurons are missing. These data argue against models in which Nkx6 proteins and Olig2 operate in a linear pathway, and instead indicate a parallel requirement for these proteins in the progression of somatic motoneuron differentiation. Thus, both visceral and somatic motoneuron differentiation appear to rely on the combined activity of cell intrinsic determinants, rather than on a single key determinant of neuronal cell fate (Pattyn, 2003).

The current analysis provides new insight also into the role of Nkx6 and Olig proteins in the generation of sMNs. Olig2 has a dual role in sMN fate determination; it suppresses the expression of Irx3 in sMN progenitors, and also promotes cell-cycle exit and neuronal differentiation by derepression of the pro-neural bHLH protein Ngn2 in the sMN progenitor domain. Nkx6 proteins are required for the expression of Olig2 in the spinal cord, and there is a similar deficit of sMNs in Nkx6 mutants, Olig2 mutants and Olig1/2 compound mutants. Because forced expression of Nkx6.1 in the chick spinal cord results in the ectopic activation of Olig2 expression and the expression of Nkx6.1 is left unaffected in Olig mutants, a model in which Olig2 acts downstream of Nkx6 proteins in the sMN pathway has been proposed. In contrast to spinal cord levels, the initial phase of Olig2 expression is unaffected in the caudal hindbrain in Nkx6 mutants, and neither the expression of Irx3 nor Nkx2.2 have encroached into the sMN progenitor domain at this stage. Despite this, all sMNs are missing. These data reveal a requirement for Nkx6.1 and Nkx6.2 in sMN fate specification that is unrelated to their role in promoting Olig2 gene expression, and further indicate that Olig2, in the absence of Nkx6 protein function, is not sufficient to specify sMN fate in the hindbrain. These findings seem to exclude the possibility that Nkx6 and Olig proteins operate in a strict linear pathway. Since both Nkx6 and Olig proteins mediate their inductive activities by acting as repressors, it appears more likely that these proteins act in parallel to exclude different sets of repressor proteins from the sMN progenitor domain. If expressed in sMN progenitors in either Nkx6 or in Olig mutant mice, such Olig2 of Nkx6 regulated repressor proteins would be predicted to act independently of each other to block sMN generation at a step downstream of Olig2. This idea gains support by the fact that forced expression of Irx3 within the sMN progenitor domain, is sufficient to inhibit sMN generation (Pattyn, 2003 and references therein).

Phox2b misexpression induces Nkx6.1 and Nkx6.2, which are expressed in branchiomotor progenitors

Within the developing vertebrate nervous system, specific subclasses of neurons are produced in vastly different numbers at defined times and locations. This implies the concomitant activation of a program that controls pan-neuronal differentiation and of a program that specifies neuronal subtype identity, but how these programs are coordinated in time and space is not well understood. Loss- and gain-of-function studies have defined Phox2b as a homeodomain transcription factor that coordinately regulates generic and type-specific neuronal properties. It is necessary and sufficient to impose differentiation towards a branchio- and viscero-motoneuronal phenotype and at the same time promote generic neuronal differentiation. The underlying genetic interactions have been examined. Phox2b has a dual action on pan-neuronal differentiation. It upregulates the expression of proneural genes (Ngn2) when expressed alone and upregulates the expression of Mash1 when expressed in combination with Nkx2.2. By a separate pathway, Phox2b represses expression of the inhibitors of neurogenesis Hes5 and Id2. The role of Phox2b in the specification of neuronal subtype identity appears to depend in part on its capacity to act as a patterning gene in the progenitor domain. Phox2b misexpression represses the Pax6 and Olig2 genes, which should inhibit a branchiomotor fate, and induces Nkx6.1 and Nkx6.2, which are expressed in branchiomotor progenitors. Phox2b behaves like a transcriptional activator in the promotion of both, generic neuronal differentiation and expression of the motoneuronal marker Islet1. These results provide insights into the mechanisms by which a homeodomain transcription factor through interaction with other factors controls both generic and type-specific features of neuronal differentiation (Dubreuil, 2002).

Zebrafish and fly Nkx6 proteins have similar CNS expression patterns and regulate motoneuron formation

Genes belonging to the Nkx, Gsh and Msx families are expressed in similar dorsovental spatial domains of the insect and vertebrate central nervous system (CNS), suggesting the bilaterian ancestor used this genetic program during CNS development. The significance of these similar expression patterns was investigated by testing whether Nkx6 proteins expressed in ventral CNS of zebrafish and flies have similar functions. In zebrafish, Nkx6.1 is expressed in early-born primary and later-born secondary motoneurons. In the absence of Nkx6.1, there are fewer secondary motoneurons and supernumerary ventral interneurons, suggesting Nkx6.1 promotes motoneuron and suppresses interneuron formation. Overexpression of fish or fly Nkx6 is sufficient to generate supernumerary motoneurons in both zebrafish and flies. These results suggest that one ancestral function of Nkx6 proteins was to promote motoneuron development (Cheesman, 2004).

Flies possess a single Nkx6 gene orthologous to vertebrate Nkx6.1 and Nkx6.2 genes. Comparing zebrafish and fly proteins, there is 93% amino acid sequence identity within the homeodomain and 80% identity within the NK decapeptide. Outside these two conserved motifs there are several other regions of high amino acid identity (Cheesman, 2004).

Zebrafish nkx6.1 transcripts are first detected at the onset of gastrulation in the embryonic shield epiblast. Near the end of gastrulation, nkx6.1 is expressed in medial neurectoderm in two wide, diffuse stripes. By the 3-4 somite stage, nkx6.1 is confined to a tight stripe in medial neural keel, extending from the midbrain through the posterior neural plate. This pattern is maintained throughout somitogenesis; Nkx6.1 expression persists in ventral spinal cord until at least 48 hpf. nkx6.1 is detected in ventral hindbrain caudal to the midbrain-hindbrain boundary and in the pancreas at later stages. At all stages examined protein and RNA patterns appear indistinguishable. Cross-sections of 24 hpf embryos reveal Nkx6.1 expression in about five longitudinal cell rows in ventral spinal cord, including both medial and lateral floorplate; thus Nkx6.1-positive cells constitute approximately the ventral third of the spinal cord. This domain is similar to the olig2 expression domain; olig2 RNA is expressed in both progenitor and postmitotic cells, thus, Nkx6.1 must also be expressed in both of these cell types. The neurectodermal stripe of nkx6.1 includes the domain in which motoneuron progenitors undergo their final division. To determine whether postmitotic motoneurons express Nkx6.1, antibody double-label experiments were performed. Islet and Nkx6.1 proteins are colocalized in PMNs during early somitogenesis (14 hpf); later Nkx6.1 protein is downregulated. By 18 hpf, Nkx6.1 and Islet proteins are largely mutually exclusive and Nkx6.1 is expressed only in a few PMNs. Cross-sections at 48 hpf reveal Nkx6.1-positive SMN nuclei surrounded by Neurolin-positive plasma membranes, indicating co-expression in these cells. Thus both PMNs and SMNs express Nkx6.1 at least transiently (Cheesman, 2004).

Recent fate-mapping has revealed that in addition to motoneurons, several types of interneurons are derived from the olig2-positive ventral spinal cord domain. Whether one of these types of interneurons express Nkx6.1 was tested by labeling individual Ventral Longitudinal Descending (VeLD) interneurons with fluorescent dextrans, immediately fixing the embryos and examining whether the labeled cells co-expressed Nkx6.1. At 20 hpf most VeLDs are Nkx6.1-positive, however by ~24 hpf VeLDs has downregulated Nkx6.1 expression. Thus, it is inferred that VeLDs express Nkx6.1 early in development but downregulate it following axon extension (Cheesman, 2004).

Studies in chick and mouse suggest Hh induces expression of Nkx6.1 in ventral neural tube (Briscoe, 2000), thus, whether Hh establishes or maintains nkx6.1 expression in zebrafish was investigated. Injection of synthetic shh mRNA causes a dramatic dorsal expansion of nkx6.1 expression. At 3-5 somites and 18 hpf, injected animals have many ectopic nkx6.1-positive cells within the neural tube. Thus, Hh is sufficient to induce nkx6.1 expression in zebrafish CNS. However, nkx6.1 expression never expanded into the dorsalmost region of the neural tube (Cheesman, 2004).

Whether Hh is necessary for nkx6.1 expression was tested. At 24 hpf, Nkx6.1 expression appeared fairly normal in syu (shh) mutants, but was greatly reduced in smu (slow muscle omitted which encodes zebrafish Smoothened) mutants. smu mutants are more severe than syu mutants, however they still retain some early Hh signaling. To further suppress Hh signaling echidna hedgehog (ehh) and tiggy winkle hedgehog (twhh) morpholino antisense oligonucleotides (MOs) were injected into syu mutants. At 24 hpf nearly 25% of injected embryos displayed severely reduced nkx6.1 neural tube expression. This probably represents the syu mutant class with the greatest loss of Hh signals. About 75% of injected embryos had weaker spinal cord nkx6.1 expression than wild types, and thus probably constituted knockdown of ehh and twhh in a wild-type or heterozygous syu background. Earlier, at 12 hpf, about 25% of a clutch of syu mutants injected with ehh and twhh MOs had dramatically reduced nkx6.1 expression. Some embryos had a more severe loss than others, but in no embryo was nkx6.1 completely absent. From these data it is inferred that Hh signals are required early to induce at least the vast majority of nkx6.1 expression and later for its maintenance. These experiments suggest that zebrafish nkx6.1 acts downstream of Hh signaling, as in chick and mouse (Cheesman, 2004).

Because in zebrafish nkx6.1 is expressed in motoneurons and their progenitors, whether nkx6.1 was sufficient to generate these cells was tested. All zebrafish primary motoneurons (PMNs) initially express islet1 (isl1); later, two specific PMNs (CaP and VaP) downregulate isl1 and initiate expression of a related gene, islet2 (isl2) whereas two other PMNs (MiP and RoP) continue to express isl1. The presence of isl1-positive or isl2-positive PMNs was assayed for by RNA in situ hybridization at 18 hpf. The isl1 probe revealed supernumerary MiPs and RoPs and the isl2 probe revealed supernumerary CaPs and VaPs. Large clusters of zn1-positive motoneurons projecting axons into the periphery were observed as compared to wild types. Many of these supernumerary PMNs were located more dorsally than native PMNs, suggesting that Nkx6.1 converts some dorsal cells to a ventral fate. Injection of a GATA-2:GFP transgenic line that expresses GFP predominantly in SMNs with synthetic nkx6.1 mRNA revealed supernumerary SMNs at 24 hpf. Because Nkx6.1 is expressed in VeLD interneurons, whether overexpression of Nkx6.1 affected this cell type was tested. VeLDs are recognized by cell body position and GABA expression. Embryos ectopically expressing nkx6.1 RNA have fewer GABA-positive VeLDs than wild types at 24 hpf, suggesting cells that would normally become VeLDs become motoneurons instead (Cheesman, 2004).

Hh can induce nkx6.1, and nkx6.1 is sufficient for formation of supernumerary PMNs and SMNs, suggesting that nkx6.1 acts downstream of Hh. This by injecting synthetic nkx6.1 mRNA into clutches of embryos derived from smu heterozygotes; smu mutants have fewer PMNs and no SMNs because of reduced Hh signaling. At 18 hpf, approximately 25% of injected embryos lack isl2 expression in caudal spinal cord, just like 25% of embryos from a smu heterozygote cross, indicating that nkx6.1 is insufficient to restore PMNs in the absence of Hh. Similarly at 30 hpf, nkx6.1 is insufficient to restore SMNs in smu mutants. It is concluded that nkx6.1 alone is sufficient to induce motoneurons only in the presence of Hh, suggesting that Nkx6.1 collaborates with additional factors downstream of Hh during motoneuron induction (Cheesman, 2004).

To test whether Nkx6.1 is required for motoneuron formation, embryos were injected with an nkx6.1-specific MO. Surprisingly, nkx6.1 MO-injected animals have normal numbers of PMNs, revealed by isl1 expression at 12 hpf and isl2 expression at 18 hpf. MO-injected embryos initiate the spontaneous tail reflex around 18 hpf, thus PMNs are functional. However, in contrast to embryos injected with a mispaired control nkx6.1 MO, which appeared wild type at 36 and 48 hpf, nkx6.1 MO-injected embryos do not swim when touched, suggesting an absence of SMNs. Consistent with this, at 48 hpf, nkx6.1 MO-injected animals consistently had fewer SMNs. At this stage there are more than 30 SMNs per spinal hemisegment; these cells are difficult to count because their somata are closely packed. Thus, MO-injected embryos were divided into two categories: those with nearly wild-type numbers of SMNs, and those with less than half of the wild-type number. Most MO-injected embryos had less than half of the wild-type number of SMNs; in a few cases SMNs were entirely absent. To test whether SMNs were dying in MO-injected embryos, TUNEL assays were performed at several stages between 18 and 48 hpf. There was no discernible difference in the number of TUNEL-positive nuclei in the ventral spinal cords of MO-injected and wild-type embryos at any stage. Whether decreased proliferation accounted for the decrease in SMNs in MO-injected embryos was also tested. BrdU incorporation showed no difference between MO-injected and wild-type embryos at 24, 30 and 36 hpf Cheesman, 2004). It is concluded that lack of SMNs is not due to a change in birth or death of these cells, suggesting that in the absence of Nkx6.1, they undergo a fate change. There are many more GABA-positive cells in the VeLD position in nkx6.1 MO-injected embryos than in wild-type embryos, suggesting that in the absence of Nkx6.1, SMNs develop as VeLD interneurons. Supporting this hypothesis is the fact that this phenotype is the opposite that of embryos overexpressing Nkx6.1 (Cheesman, 2004).

Whether Nkx6 genes have conserved functions was tested by overexpressing the fly gene in zebrafish and the zebrafish gene in flies. It was first asked whether ectopic expression of fly or zebrafish Nkx6 produces the same phenotype in zebrafish embryos. 18 hpf zebrafish embryos overexpressing fly Nkx6 mRNA had supernumerary PMNs as compared to wild types, similar to the phenotype of embryos overexpressing zebrafish nkx6.1; both fish and fly Nkx6 appear equally potent at generating ectopic PMNs in zebrafish (Cheesman, 2004).

The ability of animals to carry out their normal behavioral repertoires requires exquisitely precise matching between specific motoneuron subtypes and the muscles they innervate. However, the molecular mechanisms that regulate motoneuron subtype specification remain unclear. This study used individually identified zebrafish primary motoneurons to describe a novel role for Nkx6 and Islet1 proteins in the specification of vertebrate motoneuron subtypes. Zebrafish primary motoneurons express two related Nkx6 transcription factors. In the absence of both Nkx6 proteins, the CaP motoneuron subtype develops normally, whereas the MiP motoneuron subtype develops a more interneuron-like morphology. In the absence of Nkx6 function, MiPs exhibit normal early expression of islet1, which is required for motoneuron formation; however, they fail to maintain islet1 expression. Misexpression of islet1 RNA can compensate for loss of Nkx6 function, providing evidence that Islet1 acts downstream of Nkx6. It is suggested that Nkx6 proteins regulate MiP development at least in part by maintaining the islet1 expression that is required both to promote the MiP subtype and to suppress interneuron development (Hutchinson, 2007).

NKx6 proteins and oligodendrogenesis

In the developing spinal cord, early progenitor cells of the oligodendrocyte lineage are induced in the motor neuron progenitor (pMN) domain of the ventral neuroepithelium by the ventral midline signal Sonic hedgehog (Shh). The ventral generation of oligodendrocytes requires Nkx6-regulated expression of the bHLH gene Olig2 in this domain. In the absence of Nkx6 genes or Shh signaling, the initial expression of Olig2 in the pMN domain is completely abolished. In vivo evidence is provided for a late phase of Olig gene expression independent of Nkx6 and Shh gene activities and reveal a brief second wave of oligodendrogenesis in the dorsal spinal cord. In addition, genetic evidence is provided that oligodendrogenesis can occur in the absence of hedgehog receptor Smoothened, which is essential for all hedgehog signaling (Cai, 2005).

Nkx6.1 and kidney and pancreas development

Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells. Nkx2.2 is the member of the vertebrate homeodomain transcription factor gene family that is most homologous to the Drosophila NK2/ventral nervous system defective (vnd) gene. Nkx2.2 was originally identified as a gene that is expressed in ventral regions of the developing vertebrate CNS. In addition to Nkx2.2, five other family members have been identified in mice: Nkx2.1, Nkx2.2, Nkx2.3 and Nkx2.4 are closely related, while Nkx2.5 and Nkx2.6 represent more divergent members of the family. NK2 family members have now been shown to be key regulators of development and differentiation in several tissues: Nkx2.1 is necessary for lung, thyroid and ventral forebrain development and Nkx2.5 is required for proper heart formation. Therefore, it is possible that Nkx2.2 may play a similar role in the development of the pancreas. The endocrine pancreas is organized into clusters of cells called islets of Langerhans comprizing four well-defined cell types: alpha, beta, delta and PP cells. While recent genetic studies indicate that islet development depends on the function of an integrated network of transcription factors, the specific roles of these factors in early cell-type specification and differentiation remain elusive. Within the pancreas, Nkx2.2 is expressed in alpha, beta and PP cells, but not in delta cells. Mice homozygous for a null mutation of Nkx2.2 develop severe hyperglycemia and die shortly after birth. Immunohistochemical analysis reveals that the mutant embryos lack insulin-producing beta cells and have fewer glucagon-producing alpha cells and PP cells. Remarkably, in the mutants there remains a large population of islet cells that do not produce any of the four endocrine hormones. These cells express some beta cell markers, such as islet amyloid polypeptide and Pdx1 (a homeodomain transcription factor that is an important factor in the proliferation and differentiation of the pancreatic buds to form a mature pancreas), but lack other definitive beta cell markers including glucose transporter 2 and Nkx6.1. It is proposed that Nkx2.2 is required for the final differentiation of pancreatic beta cells, and in its absence, beta cells are trapped in an incompletely differentiated state (Sussel, 1998).

Genetic studies are beginning to outline the hierarchy of transcription factors involved in beta cell development. For example, in Nkx2.2 mutant embryos, early expression of the beta cell specific transcription factor Nkx6.1 is unaffected. However, between E12.5 and E18.5, when there are major changes taking place within the pancreas (differentiation of exocrine tissue; beta cell proliferation; delta and PP cell formation) Nkx6.1 expression disappears. The data are consistent with a model where Nkx2.2 is required for the maintenance of Nkx6.1 expression as beta cells differentiate, and that continued expression of Nkx6.1 is necessary for complete beta cell differentiation. The genetic relationship between Nkx2.2 and Pdx1 is more complicated. Early in development, Pdx1 is expressed in all cells of the pancreatic bud, and is required for expansion of the bud. However, later in embryonic development, Pdx1 becomes progressively restricted to beta cells (and some delta cells); and at approximately E14.5, Pdx1 becomes upregulated in beta cells suggesting it plays a role in beta cell differentiation. In the Nkx2.2 mutant, early expression of Pdx1 is not affected. The later beta cell restriction of Pdx1 expression also occurs, but is quantitatively reduced in comparison to wild-type beta cells. Therefore, Nkx2.2 may be required for inducing high level expression of Pdx1 in beta cells, and the up-regulation of Pdx1 may be a necessary step in the final differentiation of the beta cell. In contrast to Nkx6.1 or Pdx1 expression, the expression of Isl1, Pax6 and Brn4 during embryogenesis does not appear to require Nkx2.2. These genes may therefore either lie upstream or in different pathways relative to the Nkx genes. Since Isl1 is expressed in islet cells soon after they exit the cell cycle, normal expression of Isl1 in the Nkx2.2 mutant suggests that all the islet cells are able to normally exit a proliferating state and proceed with a program of differentiation. This result supports the hypothesis that the immature beta cells are able to initiate beta cell development and it is subsequent steps of terminal differentiation that are blocked (Sussel, 1998 and references).

beta-Cell differentiation factor Nkx6.1 is a homeodomain protein expressed in developing and mature beta-cells in the pancreatic islets of Langerhans. To understand how it contributes to beta-cell development and function, its DNA binding and transactivation properties were characterized. A single copy of the homeodomain of Nkx6. 1 binds to a strictly conserved 8-base pair DNA consensus sequence, TTAATTAC; even minor variations to this consensus reduce DNA binding affinity significantly. Full-length Nkx6.1, however, has markedly reduced DNA binding affinity due to an acidic domain at the carboxyl end of the molecule that functions as a mobile binding interference domain capable of interrupting the interaction between DNA and DNA binding domains of the helix-turn-helix type. When expressed in fibroblast cell lines, Nkx6.1 represses transcription through isolated Nkx6.1 binding sites; in beta-cell lines, Nkx6.1 specifically represses the intact insulin promoter through TAAT-containing sequences. In Gal4 one-hybrid fusion studies, transcriptional repression maps to a discreet region within the amino terminus. These findings suggest a model in which Nkx6.1, regulated by interactions through its carboxyl terminus, directs the repression of specific genes in developing and mature beta-cells (Mirmira, 2000).

Most insulin-producing beta-cells in the fetal mouse pancreas arise during the secondary transition, a wave of differentiation starting at embryonic day 13. Disruption of homeobox gene Nkx6.1 in mice leads to loss of beta-cell precursors and blocks beta-cell neogenesis specifically during the secondary transition. In contrast, islet development in Nkx6.1/Nkx2.2 double mutant embryos is identical to Nkx2.2 single mutant islet development: beta-cell precursors survive but fail to differentiate into beta-cells throughout development. Together, these experiments reveal two independently controlled pathways for beta-cell differentiation, and place Nkx6.1 downstream of Nkx2.2 in the major pathway of beta-cell differentiation (Sander, 2000b).

In the mature pancreas, the homeodomain transcription factor Nkx6.1 is uniquely restricted to beta-cells. Nkx6.1 also is expressed in developing beta-cells and plays an essential role in their differentiation. Among cell lines, both beta- and alpha-cell lines express nkx6.1 mRNA; but no protein can be detected in the alpha-cell lines, suggesting that post-transcriptional regulation contributes to the restriction of Nkx6.1 to beta-cells. To investigate the regulator of Nkx6.1 expression, the promoter structure of the mouse nkx6.1 gene has been analyzed, and regions that direct cell type-specific expression were identified. The nkx6.1 gene has a long 5'-untranslated region (5'-UTR) downstream of a cluster of transcription start sites. nkx6.1 gene sequences from -5.6 to +1.0 kilobase pairs have specific promoter activity in beta-cell lines but not in NIH3T3 cells. This activity is dependent on sequences located at about -800 base pairs and on the 5'-UTR. Electrophoretic mobility shift assays demonstrate that homeodomain transcription factors PDX1 and Nkx2.2 can bind to the sequence element located at -800 base pairs. In addition, dicistronic assays establish that the 5'-UTR region functions as a potent internal ribosomal entry site, providing cell type-specific regulation of translation. These data demonstrate that complex regulation of both Nkx6.1 transcription and translation provides the specificity of expression required during pancreas development (Watada, 2000).

In the pancreas, the NK homeodomain transcription factor Nkx6.1 is required for the development of beta-cells and is believed to function as a potent repressor of transcription upon binding to A/T-rich sequences within the promoter region of target genes. Because the nkx6.1 promoter itself contains several such sequences, the possibility is considered that the expression level and restricted pattern of the nkx6.1 gene might be precisely regulated by one or more homeodomain transcription factors, including Nkx6.1 itself. In this report, a novel beta-cell-specific enhancer element is identified in the nkx6.1 gene between -157 and -30 bp (relative to the transcriptional start site) that harbors a conserved A/T-containing sequence flanked by G/C-rich stretches. Although the islet homeodomain-containing activator Pdx-1 is unable to stimulate transcription of a reporter gene through this enhancer element in mammalian cell lines, strikingly, Nkx6.1 robustly activates transcription through direct interaction with the A/T-rich sequence in this element. This activation is indeed transcriptional in nature (and not secondary to translational effects) and is mediated by a modular acidic sequence within the COOH-terminal domain of Nkx6.1. It has been shown by EMSAs that Nkx6.1 binds to the beta-cell-specific enhancer in vitro; by chromatin immunoprecipitation assays it has been shown that Nkx6.1 natively occupies this region in vivo in betaTC3 cells. It is therefore concluded that Nkx6.1 is a bifunctional transcription factor that serves to maintain the specific expression of its own gene during beta-cell differentiation while simultaneously effecting broader gene repression events (Iype, 2004).

In diabetic individuals, the imbalance in glucose homeostasis is caused by loss or dysfunction of insulin-secreting ß-cells of the pancreatic islets. As successful generation of insulin-producing cells in vitro could constitute a cure for diabetes, recent studies have explored the molecular program that underlies ß-cell formation. From these studies, the homeodomain transcription factor NKX6.1 has proven to be a key player. In Nkx6.1 mutants, ß-cell numbers are selectively reduced, while other islet cell types develop normally. However, the molecular events downstream of NKX6.1, as well as the molecular pathways that ensure residual ß-cell formation in the absence of NKX6.1 have remained largely unknown. This study shows that the Nkx6.1 paralog, Nkx6.2, is expressed during pancreas development and partially compensates for NKX6.1 function. Surprisingly, analysis of Nkx6 compound mutant mice reveals a previously unrecognized requirement for NKX6 activity in alpha-cell formation. This finding suggests a more general role for NKX6 factors in endocrine cell differentiation than formerly suggested. Similar to NKX6 factors, the transcription factor MYT1 has recently been shown to regulate alpha- as well as ß-cell development. Expression of Myt1 depends on overall Nkx6 gene dose, and therefore identifies Myt1 as a possible downstream target of Nkx6 genes in the endocrine differentiation pathway (Henseleit, 2005).

Despite much progress in identifying transcriptional regulators that control the specification of the different pancreatic endocrine cell types, the spatiotemporal aspects of endocrine subtype specification have remained largely elusive. This study addressed the mechanism by which the transcription factors Nkx6.1 (Nkx6-1) and Nkx6.2 (Nkx6-2) orchestrate development of the endocrine alpha- and beta-cell lineages. Specifically, an assay was performed for the rescue of insulin-producing beta-cells in Nkx6.1 mutant mice upon restoring Nkx6 activity in select progenitor cell populations with different Nkx6-expressing transgenes. Beta-cell formation and maturation was restored when Nkx6.1 was expressed in multipotential Pdx1+ pancreatic progenitors, whereas no rescue was observed upon expression in committed Ngn3+ (Neurog3+) endocrine progenitors. Although not excluding additional roles downstream of Ngn3, this finding suggests a first requirement for Nkx6.1 in specifying beta-cell progenitors prior to Ngn3 activation. Surprisingly, although Nkx6.2 only compensates for Nkx6.1 in alpha-but not in beta-cell development in Nkx6.1-/- mice, a Pdx1-promoter-driven Nkx6.2 transgene had the same ability to rescue beta-cells as the Pdx1-Nkx6.1 transgene. This demonstrates that the distinct requirements for Nkx6.1 and Nkx6.2 in endocrine differentiation are a consequence of their divergent spatiotemporal expression domains rather than their biochemical activities and implies that both Nkx6.1 and Nkx6.2 possess alpha- and beta-cell-specifying activities (Nelson, 2007). Search=20627083">20627083

The molecular mechanisms that underlie cell lineage diversification of multipotent progenitors in the pancreas are virtually unknown. This study shows that the early fate choice of pancreatic progenitors between the endocrine and acinar cell lineage is restricted by cross-repressive interactions between the transcription factors Nkx6.1/Nkx6.2 (Nkx6) and Ptf1a. Using genetic loss- and gain-of-function approaches, it was demonstrated that Nkx6 factors and Ptf1a are required and sufficient to repress the alternative lineage program and to specify progenitors toward an endocrine or acinar fate, respectively. The Nkx6/Ptf1a switch only operates during a critical competence window when progenitors are still multipotent and can be uncoupled from cell differentiation. Thus, cross-antagonism between Nkx6 and Ptf1a in multipotent progenitors governs the equilibrium between endocrine and acinar cell neogenesis required for normal pancreas development (Schaffer, 2010).

Nkx6-1 controls the identity and fate of red nucleus and oculomotor neurons in the mouse midbrain

Little is known about the cues controlling the generation of motoneuron populations in the mammalian ventral midbrain. This study shows that Otx2 provides the crucial anterior-posterior positional information for the generation of red nucleus neurons in the murine midbrain. Moreover, the homeodomain transcription factor Nkx6-1 controls the proper development of the red nucleus and of the oculomotor and trochlear nucleus neurons. Nkx6-1 is expressed in ventral midbrain progenitors and acts as a fate determinant of the Brn3a+ (also known as Pou4f1) red nucleus neurons. These progenitors are partially dorsalized in the absence of Nkx6-1, and a fraction of their postmitotic offspring adopts an alternative cell fate, as revealed by the activation of Dbx1 and Otx2 in these cells. Nkx6-1 is also expressed in postmitotic Isl1+ oculomotor and trochlear neurons. Similar to hindbrain visceral (branchio-) motoneurons, Nkx6-1 controls the proper migration and axon outgrowth of these neurons by regulating the expression of at least three axon guidance/neuronal migration molecules. Based on these findings, additional evidence is provided that the developmental mechanism of the oculomotor and trochlear neurons exhibits more similarity with that of special visceral motoneurons than with that controlling the generation of somatic motoneurons located in the murine caudal hindbrain and spinal cord (Prakash, 2009).

During mouse development, distinct neuronal populations organized in discrete nuclei arise in the ventral midbrain (VM). The best characterized are the substantia nigra and ventral tegmental area dopaminergic (mDA) neurons due to their clinical relevance, but less is known about the cues controlling the development of other nuclei in the VM or rostral hindbrain. These include the oculomotor nucleus (OM), the trochlear nucleus (TN) and the red nucleus (RN). The OM and TN give rise to the third (nIII) and fourth (nIV) cranial nerve, respectively. The nIII innervates the ipsilateral extraocular muscles and ciliary ganglion, thereby controlling most eye movements, eye accommodation and pupil contraction, and the nIV regulates the movements of the contralateral superior oblique muscle of the eye. The RN is located in close vicinity to the OM and mDA area and contains both excitatory glutamate- and inhibitory {gamma}-aminobutyric acid (GABA)-synthesizing neurons, which project to the cerebellum, brainstem and spinal cord. Together with the corticospinal tract, the rubrospinal tract plays a fundamental role in the control of limb movements (Prakash, 2009).

Whereas little is known about the molecular mechanism and factors controlling the specification of the RN, similar cues for the motoneuron (MN) and interneuron (IN) populations arising in the vertebrate hindbrain and spinal cord appear to act during the development of the OM and TN. In particular, the POU homeodomain (HD) transcription factor (TF) Pou4f1 (also known as Brn3a) is expressed in postmitotic RN neurons and is required for their survival. A similar RN phenotype has been reported in mouse mutants lacking the homeobox gene Emx2. The LIM HD TF islet1 (Isl1), a generic MN marker, is expressed in the OM/TN and, although Isl1 is necessary for survival of spinal MNs, its function in OM/TN development remains unknown. The paired-like homeobox gene Phox2a is expressed in OM/TN progenitors and, together with its paralog Phox2b, in their postmitotic offspring. The OM and TN are lost in Phox2a-/- but not in Phox2b-/- mice, indicating that Phox2a is a crucial fate determinant in OM/TN precursors (Prakash, 2009).

The induction of fate-determining TFs by extrinsic and intrinsic signals along the dorsoventral (D/V) and anterior-posterior (A/P) axis of the neural tube is a crucial process that is likely to precede the differentiation of RN and OM/TN neurons in the VM/rostral hindbrain. Relevant studies support a general model in which the glycoprotein sonic hedgehog (Shh), secreted from the ventral midline of the neural tube (the floor plate, FP), is essential for D/V patterning by controlling the expression of different sets of HD TFs. Among these, the HD TFs Nkx6-1 and Nkx6-2 are transcriptional repressors induced by Shh signaling in the ventral hindbrain and spinal cord, and are required for the specification of hindbrain and spinal somatic MNs (sMNs) and INs, as well as for proper muscle nerve formation and innervation patterns. Moreover, Nkx6-1 and Nkx6-2 are also required for the proper migration and axon pathfinding of hindbrain visceral (branchio-) MNs (vMNs), but not for their initial specification. Ablation of Nkx6 function leads to the derepression of the Shh-repressed HD TFs developing brain homeobox 1 and 2 (Dbx1 and Dbx2) in the ventral spinal cord, and to a fate-switch of MNs and ventral (V2) INs into more dorsal (V0) INs. Notably, most of these studies have been focussed on spinal cord and hindbrain, thus leaving largely unknown the role of Nkx6-1 and Nkx6-2 in the development of VM neuronal populations (Prakash, 2009).

The mammalian HD protein Otx2 controls fore- and mid-brain patterning. Conditional inactivation of Otx2 in the mid-/hind-brain region of the mouse results in a complete loss of the RN and hypoplasia of the OM. This paper reports a study the functional involvement of Otx2 and Nkx6-1 in specification of identity and fate of RN and OM/TN neuronal populations. It shows that Otx2 is necessary and sufficient for the maintenance and ectopic induction of Nkx6-1 in the VM/rostral hindbrain of mutant mice. Moreover, Nkx6-1 is required for the generation of Pou4f1+ RN neurons and for the proper migration and axon outgrowth of OM/TN neurons, thus revealing intriguing similarities between the midbrain phenotype of Nkx6-1-/- mice and the sMN and vMN defects previously described in the same mutants. These findings suggest that functional aspects of the mechanism controlling hindbrain and spinal cord MN development have been conserved and recruited to a more rostral position in the mouse brain (Prakash, 2009).


Search PubMed for articles about Drosophila Nkx6/HGTX

Arber, S., Han, B., Mendelsohn, M., Smith, M., Jessell, T. M. and Sockanathan, S. (1999). Requirement for the homeobox gene Hb9 in the consolidation of motor neuron identity. Neuron 23: 659-674. 10482234

Briscoe, J., Pierani, A., Jessell, T. M. and Ericson, J. (2000). A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101: 435-445. 10830170

Broihier, H. T. and Skeath, J. B. (2002). Drosophila homeodomain protein dHb9 directs neuronal fate via crossrepressive and cell-nonautonomous mechanisms Neuron 35: 39-50. 12123607

Broihier, H. T., Kuzin, A., Zhu, Y., Odenwald, W. and Skeath, J. B. (2004). Drosophila homeodomain protein Nkx6 coordinates motoneuron subtype identity and axonogenesis. Development 131: 5233-5242. 15456721

Brody, T. and Odenwald, W. F. (2000). Programmed transformations in neuroblast gene expression during Drosophila CNS lineage development. Developmental Biology 226: 34-44.

Cai, J., Qi, Y., Wu. R., Moddeman, G., Fu, H., Liu, R. and Qiu, M. (2001). Mice lacking the nkx6.2 (gtx) homeodomain transcription factor develop and reproduce normally. Mol. Cell. Biol. 21: 4399-4403. 11390667

Cai, J., et al. (2005). Generation of oligodendrocyte precursor cells from mouse dorsal spinal cord independent of Nkx6 regulation and Shh signaling. Neuron 45: 41-53. 15629701

Chang, S., Johnston, R. J. and Hobert O. (2003). A transcriptional regulatory cascade that controls left/right asymmetry in chemosensory neurons of C. elegans. Genes Dev. 17(17): 2123-37. 12952888

Cheesman, S. E., Layden, M. J., Von Ohlen, T., Doe, C. Q. and Eisen, J. S. (2004). Zebrafish and fly Nkx6 proteins have similar CNS expression patterns and regulate motoneuron formation. Development 131(21): 5221-32. 15456722

Denes, A. S., et al. (2007). Molecular architecture of annelid nerve cord supports common origin of nervous system centralization in bilateria. Cell 129(2): 277-88. Medline abstract: 17448990

Dubreuil, V., et al. (2002). The role of Phox2b in synchronizing pan-neuronal and type-specific aspects of neurogenesis. Development 129: 5241-5253. 12399315

Evans, T. A., Bashaw, G. J. (2010) Functional diversity of Robo receptor immunoglobulin domains promotes distinct axon guidance decisions. Curr Biol 20: 567-572. PubMed ID: 20206526

Henseleit, K. D., et al. (2005). NKX6 transcription factor activity is required for alpha- and beta-cell development in the pancreas. Development 132(13): 3139-49. 15944193

Hutchinson, S. A., Cheesman, S. E., Hale, L. A., Boone, J. Q. and Eisen, J. S. (2007). Nkx6 proteins specify one zebrafish primary motoneuron subtype by regulating late islet1 expression. Development. 134(9): 1671-7. Medline abstract: 17376808

Isshiki, T., Pearson, B., Holbrook, S. and Doe, C. Q. (2001). Drosophila neuroblasts sequentially express transcription factors which specify the temporal identity of their neuronal progeny. Cell 106: 511-521. 11525736

Iype, T., et al. (2004), The transcriptional repressor Nkx6.1 also functions as a deoxyribonucleic acid context-dependent transcriptional activator during pancreatic beta-cell differentiation: evidence for feedback activation of the nkx6.1 gene by Nkx6.1. Mol. Endocrinol. 18(6): 1363-75. 15056733

Johnston, R. J. and Hobert, O. (2003). A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans. Nature 426(6968): 845-9. 14685240

Kambadur, R., Koizumi, K., Stivers, C., Nagle, J., Poole, S. J. and Odenwald, W. F. (1998). Regulation of POU genes by castor and hunchback establishes layered compartments in the Drosophila CNS. Genes Dev. 12: 246-260. 9436984

Landgraf, M., Roy, S., Prokop, A., VijayRaghavan, K. and Bate, M. (1999). even-skipped determines the dorsal growth of motor axons in Drosophila. Neuron 22: 43-52. 10027288

Liu, R., et al. (2003). Region-specific and stage-dependent regulation of Olig gene expression and oligodendrogenesis by Nkx6.1 homeodomain transcription factor. Development 130(25): 6221-31. 14602683

Marques, G., et al. (2002). The Drosophila BMP type II receptor Wishful thinking regulates neuromuscular synapse morphology and function. Neuron 33: 529-543. 11856528

Mirmira, R. G., Watada, H. and German, M. S. (2000). Beta-cell differentiation factor Nkx6.1 contains distinct DNA binding interference and transcriptional repression domains. J. Biol. Chem. 275(19): 14743-51. 10799563

Muhr, J., et al. (2001). Groucho-mediated transcriptional repression establishes progenitor cell pattern and neuronal fate in the ventral neural tube. Cell 104: 861-873. 11290324

Müller, M., Jabs, N., Lorke, D. E., Fritsch, B. and Sander, M. (2003). Nkx6.1 controls migration and axon pathfinding of cranial branchio-motoneurons. Development 130: 5815-5826. 14534138

Nelson, S. B., Schaffer, A. E. and Sander, M. (2007). The transcription factors Nkx6.1 and Nkx6.2 possess equivalent activities in promoting beta-cell fate specification in Pdx1+ pancreatic progenitor cells. Development 134: 2491-2500. Medline abstract: 17537793

Odden, J. P., Holbrook, S. and Doe, C. Q. (2002). Drosophila Hb9 is expressed in a subset of motoneurons and interneurons where it regulates gene expression and axon pathfinding. J. Neurosci. 22: 9143-914. 12417636

Palmer, R. E., Inoue, T., Sherwood, D. R., Jiang, L. I. and Sternberg, P. W. (2002). Caenorhabditis elegans cog-1 locus encodes GTX/Nkx6.1 homeodomain proteins and regulates multiple aspects of reproductive system development. Dev. Biol. 252(2): 202-13. 12482710

Pattyn, A., Vallstedt, A., Dias, J. M., Sander, M. and Ericson, J. (2003). Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain. Development 130: 4149-4159. 12874134

Prakash, N., et al. (2009). Nkx6-1 controls the identity and fate of red nucleus and oculomotor neurons in the mouse midbrain. Development 136(15): 2545-55. PubMed Citation: 19592574

Qiu, M., Shimamura, K., Sussel, L., Chen, S. and Rubenstein, J. L. (1998). Control of anteroposterior and dorsoventral domains of Nkx-6.1 gene expression relative to other Nkx genes during vertebrate CNS development. Mech. Dev. 72: 77-88. 9533954

Sander, M., Paydar, S., Ericson, J., Briscoe, J., Berber, E., German, M., Jessell, T. M. and Rubenstein, J. L. R. (2000a). Ventral neural patterning by Nkx homeobox genes: Nkx6.1 controls somatic motor neuron fates and ventral interneuron fates. Genes Dev. 14: 2134-2139. 10970877

Sander, M., et al. (2000b). Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas. Development 127: 5533-5540. PubMed Citation: 11076772

Santiago, C., Labrador, J. P., Bashaw, G. J. (2014) The Homeodomain Transcription Factor Hb9 Controls Axon Guidance in Drosophila through the Regulation of Robo Receptors. Cell Rep 7: 153-165. PubMed ID: 24685136

Schaffer, A. E., et al. (2010). Nkx6 transcription factors and Ptf1a function as antagonistic lineage determinants in multipotent pancreatic progenitors. Dev. Cell 18(6): 1022-9. PubMed Citation: 19906861

Seroka, A., Yazejian, R. M., Lai, S. L. and Doe, C. Q. (2020). A novel temporal identity window generates alternating Eve(+)/Nkx6(+) motor neuron subtypes in a single progenitor lineage. Neural Dev 15(1): 9. PubMed ID: 32723364

Spitzweck, B., Brankatschk, M., Dickson, B. J. (2010) Distinct protein domains and expression patterns confer divergent axon guidance functions for Drosophila Robo receptors. Cell 140: 409-420. PubMed ID: 20144763

Sussel, L., et al. (1998). Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells. Development 125(12): 2213-2221. PubMed Citation: 9584121

Syu, L. J., Uhler, J., Zhang, H. and Mellerick, D. M. (2009). The Drosophila Nkx6 homeodomain protein has both activation and repression domains and can activate target gene expression. Brain Res. 1266: 8-17. PubMed Citation: 19232326

Thaler, J., et al. (1999). Active suppression of interneuron programs within developing motor neurons revealed by analysis of homeodomain factor HB9. Neuron 23(4): 675-87. 10482235

Thor, S. and Thomas, J. B. (1997). The Drosophila islet gene governs axon pathfinding and neurotransmitter identity. Neuron 18: 397-409. 9115734

Thor, S., Andersson, S. G., Tomlinson, A. and Thomas, J. B. (1999). A LIM-homeodomain combinatorial code for motor-neuron pathway selection. Nature 397: 76-80. 9892357

Uhler, J., Garbern J., Yang, L., Kamholz, J. and Mellerick, D. M. (2002). Nkx6, a novel Drosophila homeobox gene regulated by vnd. Mech. Dev. 116: 105-116. 12128210

Vallstedt, A., Muhr, J., Pattyn, A., Pierani, A., Mendelsohn, M., Sander, M., Jessell, T. M. and Ericson, J. (2001). Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneuron specification. Neuron 31: 743-755. 11567614

Watada, H., Mirmira, R. G., Leung, J. and German, M. S. (2000). Transcriptional and translational regulation of beta-cell differentiation factor Nkx6.1. J. Biol. Chem. 275(44): 34224-30. 10938085

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