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).
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),
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).
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