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),
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),
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date revised: 30 March 2008
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