1 Department of Genetics, Washington University School of Medicine, 4566 Scott
Avenue, St Louis, MO 63110, USA
2 Neural Cell-Fate Determinants Section, NINDS, NIH, 36 Convent Drive MSC 4130,
Bethesda, MD 20892-4130, USA
Author for correspondence (e-mail:
heather.broihier{at}case.edu)
Accepted 11 August 2004
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SUMMARY |
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Key words: Drosophila melanogaster, Neuronal fate specification, Motoneurons, Interneurons, Axon outgrowth, Nkx6, hb9 (exex), lim3, islet, eve, vnd
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Introduction |
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In many model systems, MNs that extend axons along common trajectories
express similar sets of transcriptional regulators which in turn
regulate key aspects of the differentiation of these MN subtypes (for a
review, see Thor and Thomas,
2002). Drosophila MNs are classified by the location of
the body wall muscles they innervate. MNs that innervate dorsal body wall
muscles in Drosophila express the homeodomain (HD) transcription
factor Even-skipped (Eve) (Doe et al.,
1988
). Furthermore, genetic analyses indicate that Eve is a key
determinant of the fate of dorsally projecting MNs
(Landgraf et al., 1999
). Eve
engages in a cross-repressive interaction with the HD protein Hb9, a
determinant of ventrally projecting MNs
(Broihier and Skeath, 2002
).
Ventrally projecting MNs also express the HD transcription factors Lim3 and
Islet. Functional analyses have demonstrated that these three HD factors are
required for proper axon guidance of ventrally projecting MNs
(Broihier and Skeath, 2002
;
Odden et al., 2002
;
Thor and Thomas, 1997
;
Thor et al., 1999
). The
genetic hierarchy governing the fate of ventrally projecting neurons has,
however, remained elusive as Lim3, Islet, and Hb9 are expressed independently
of each other.
Lim3, Islet, and Hb9 are conserved regulators of MN cell fate whose
vertebrate homologs Lhx3/4, Islet 1/2, and Hb9 play key roles
in vertebrate MN specification (Arber et
al., 1999; Sharma et al.,
1998
; Thaler et al.,
1999
; Tsuchida et al.,
1994
). In vertebrates, the genetic hierarchy linking the three
transcription factors appears more linear than in Drosophila, as Hb9
regulates Lhx3/4 and Isl1/2 expression
(Arber et al., 1999
;
Thaler et al., 1999
). As in
Drosophila, the vertebrate Eve homolog, Evx1, is expressed in a
distinct population of neurons in this case, a subset of vertebrate
interneurons (Moran-Rivard et al.,
2001
).
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 (Briscoe et al., 2000).
For example, the pair of Nkx-class HD proteins, Nkx6.1 and Nkx6.2 (Nkx6
proteins), have complementary expression patterns in MN and interneuron
progenitors (Briscoe et al.,
2000
; Cai et al., 1999). Nkx6.1/Nkx6.2 compound mutants exhibit a
near complete loss of somatic MNs, demonstrating that Nkx6 proteins are
essential for MN generation (Sander et
al., 2000
; Vallstedt et al.,
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 et al.,
2003
; Pattyn et al.,
2003
).
To explore further the genetic networks behind neuronal diversification in Drosophila, we investigated the role of the Drosophila Nkx6 homolog in regulating distinct MN fates. We characterized genetic interactions between Nkx6 and factors essential for neuronal fate acquisition. We present evidence that Nkx6 collaborates with hb9 (exex FlyBase) to regulate the fate of distinct neuronal populations. Our 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, we demonstrate that 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.
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Materials and methods |
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Nkx6 cDNA and UAS-Nkx6
We isolated a full-length Nkx6 cDNA via RT-PCR from RNA prepared
from a 0- to 20-hour collection of Oregon R embryos. polyA RNA was
prepared using the RNeasy midi kit and oligotex beads (Qiagen) and converted
into cDNA using superscript II reverse transcriptase (Gibco BRL).
Nkx6 cDNA was generated using primers that amplify from the predicted
start to the predicted stop codon. We cloned and sequenced the 1539 bp
product, which matches Nkx6 cloned from an embryonic library
(Uhler et al., 2002) at the
amino acid level. To create UAS-Nkx6, we inserted the Nkx6
cDNA into the Not1 site of pUAST
(Brand and Perrimon, 1993
) and
created germline transformants following standard methods.
Antibody production, immunofluorescent, and immunohistochemical studies
Amino acids 34-386 of Nkx6 were cloned into pET29a (Novagen) for protein
expression and purification. This antigen was used to immunize rats at Pocono
Rabbit Farm. Confirming antibody specificity, we failed to detect Nkx6 protein
in embryos homozygous for Nkx6D25 or Df(3L)fz-D21 (data
not shown). The following primary antibodies were used: rat anti-Islet, guinea
pig anti-Lim3, rabbit anti-Hb9 (Broihier
and Skeath, 2002); rabbit anti-Odd (E. Ward); rabbit anti-Vnd (D.
Mellerick); rabbit anti-GFP (P. Silver); rabbit anti-Eve (M. Frasch); mouse
anti-Eve (N. Patel); mouse anti-Myc (Sigma); rabbit anti-ßgal (ICN; mouse
anti-ßgal (Promega); and mouse monoclonal 1D4/Fas2 and 7G10/Fas3 were
generated by C. Goodman's Laboratory and obtained from the Developmental
Studies Hybridoma Bank. The anti-Lim3 antibody was affinity purified using the
Ultralink Immobilization Kit (Pierce). We used the Vector ABC kit for
immunohistochemistry and Alexa-488 and Alexa-594 with appropriate species
specificity for immunofluorescence (Molecular Probes).
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Results |
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To establish the identity of Nkx6-positive neurons, we compared Nkx6
expression to markers of defined neuronal subsets. We first investigated
whether Nkx6 is expressed in MN and interneuron populations in
Drosophila. We compared Nkx6 expression to that of Odd-skipped (Odd),
and find that Nkx6 and Odd are co-expressed in the MP1 and dMP2 interneurons
(data not shown). We then asked whether Nkx6 is present in distinct MN groups.
To this end, we compared Nkx6 expression to that of Hb9 and Eve. Hb9 is
expressed in ventrally and laterally projecting MNs while Eve is expressed in
dorsally projecting MNs (Broihier and
Skeath, 2002; Landgraf et al.,
1999
; Odden et al.,
2002
). Like Hb9 and Eve, Nkx6 and Eve are also expressed in
complementary patterns (Fig.
1F). On the other hand, the majority of Nkx6-expressing cells
express Hb9, although Nkx6 is expressed in slightly more neurons than Hb9
(Fig. 1H-J). The extensive
co-expression of Nkx6 and Hb9 suggested that Nkx6 is also expressed in
ventrally projecting MNs. Confirming this, we find that Nkx6 is co-expressed
with a Lim3-
myc transgene, a marker of RP 1,3,4,5 (RP MNs) a
group of well-characterized ventrally projecting MNs (arrowheads in
Fig. 1G)
(Thor et al., 1999
). This
analysis established that Nkx6 is expressed in both interneurons and ventrally
projecting MNs.
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 et
al., 2000; Vallstedt et al.,
2001
). However, we find that Nkx6 and Hb9 are expressed
independently of each other in the Drosophila CNS (data not shown).
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.
Nkx6 and hb9 act in parallel to repress vnd
During early CNS development, Nkx6 is co-expressed with Ventral nervous
system defective (Vnd) in a subset of medial column NBs (see Fig. S2 in the
supplementary material), prompting us to investigate the genetic relationship
between vnd and Nkx6. Vnd expression marks medial column CNS
NBs and is required for the development of these cells
(Chu et al., 1998;
Jimenez and Campos-Ortega,
1990
; Skeath et al.,
1994
). We first compared Nkx6 and Vnd expression 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
(Fig. 2A-C). At stage 9, Nkx6
is expressed in CNS midline precursors, while Vnd is expressed in ventral
neuroectoderm flanking the midline (Fig.
2A). 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 (Fig. 2B). The
complementary patterns of Nkx6 and Vnd in GMCs and neurons are maintained
throughout embryogenesis (Fig.
2C). These data raised the possibility that opposing activities of
Nkx6 and vnd help establish and maintain their respective
expression patterns.
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In the reciprocal experiment, we found that postmitotic misexpression of Nkx6 dramatically reduces the number of Vnd-positive neurons (compare Fig. 2F,G). Normally, 10.0±1.3 neurons express Vnd per hemisegment (n=30) 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 (data not shown). Thus, Nkx6 is sufficient but not necessary to repress vnd expression.
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 we 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 (2.8±1.7 neurons per hemisegment; n=48; Fig. 2H) while hb9 mutants exhibit wild-type Vnd expression (data not shown). However, removal of both hb9 and Nkx6 leads to an overproduction of Vnd-positive neurons as 13.6±2.1 Vnd-positive neurons (n=41) develop in double mutant embryos relative to ten in wild type (compare Fig. 2F and 2I). 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.
Nkx6 and hb9 collaborate to regulate MN fate
We next explored the regulatory relationship between Nkx6 and
hb9 both of which are expressed in ventrally projecting
motoneurons and the dorsal motoneuron determinant eve. eve
and hb9 engage in a cross-repressive relationship to maintain their
expression in distinct neuronal populations
(Broihier and Skeath, 2002).
Since Nkx6 and Eve are also expressed in non-overlapping populations of
neurons (Fig. 1E), we asked
whether they repress each other. We 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 (compare
Fig. 3A and 3B), demonstrating
that eve is sufficient to repress Nkx6.
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We find, however, that 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 (n=58;
Fig. 3E). This number
represents an increase of two Eve-positive neurons relative to wild type
(Broihier and Skeath, 2002).
Significantly, stage 15 hb9KK30 Nkx6D25 double
mutant embryos display 24.0±3.7 Eve-positive neurons per hemisegment
(n=50; Fig. 3F),
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 (see Discussion). To confirm this phenotype is
caused by loss of Nkx6 activity from an hb9 mutant
background, we injected double-stranded Nkx6 RNA into
hb9KK30 mutant embryos. We find an average of
24.8±5.9 Eve-positive neurons per hemisegment in these embryos
(n=39; see Fig. S1 in the supplementary material), 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. We next
investigated whether hb9 and Nkx6 coordinate the
specification of ventrally projecting MN identity. 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
(Fig. 1F,
Fig. 6A)
(Sink and Whitington, 1991a;
Sink and Whitington, 1991b
;
Schmid et al., 1999
). Since
both Nkx6 and Hb9 are expressed in RP1,3,4,5
(Fig. 1G) (Broihier and Skeath, 2002
), we
asked whether these neurons develop properly in hb9 Nkx6 double
mutant embryos. Islet and Lim3 are markers of RP1,3,4,5 identity
(Thor and Thomas, 1997
;
Thor et al., 1999
) and are
expressed in the these MNs in embryos singly mutant for Nkx6 or
hb9 (data not shown) (Broihier and
Skeath, 2002
). However, expression of Islet and Lim3 in the
RP1,3,4,5 MNs is strongly reduced in hb9 Nkx6 double mutant embryos
(arrowheads in Fig. 3G-J).
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,
we favor the model that Nkx6 and hb9 promote ventrally
projecting MN identity by repressing eve expression in RP MNs (see
Discussion).
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To ensure that loss of Nkx6 activity is responsible for the
observed axonal phenotypes, we assayed whether Nkx6 misexpression in
a Nkx6 mutant background rescues the ISNb outgrowth phenotype. We
used targeted transposition to engineer an Nkx6GAL4
enhancer trap from the Nkx6P[JG(LacZ)] enhancer trap
(Materials and methods) (Sepp and Auld,
1999). We then used the Nkx6GAL4 driver to
express Nkx6 in Nkx6GAL4/Nkx6D25
mutant embryos (Fig. 4E). We
find that Nkx6 expression is sufficient to rescue ISNb outgrowth in
72% of hemisegments (n=154), compared to 27% in the absence of
Nkx6 misexpression (n=166). 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.
Since Nkx6 and hb9 act in parallel to regulate neuronal fate (see above), we wondered 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 (Fig. 4E). Therefore, while Nkx6 and hb9 collaborate to regulate multiple neuronal fates, Nkx6 plays a specific non-redundant role to promote axonogenesis.
To examine the role of Nkx6 during axonogenesis in more detail, we focused 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 (Fig. 4F). However, we fail to detect Hb9-positive axons in ISNb in Nkx6D25 homozygous mutant embryos (Fig. 4G). 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, we followed Hb9-positive axons in the nerve cords of Nkx6 mutant embryos. In wild type, Hb9-positive interneurons extend axons in multiple longitudinal fascicles in the CNS (Fig. 4H). In contrast, in Nkx6 mutant embryos, we observe very few Hb9-positive axons projecting along longitudinal fascicles (Fig. 4I). 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.
Nkx6 overexpression leads to ISNb overgrowth
The impaired axon extension observed in Nkx6 mutant embryos argues
that Nkx6 is a positive mediator of axon growth. To test this model,
we analyzed axon growth in embryos that over-expressed Nkx6 in all
postmitotic neurons. To ensure high levels of Nkx6 expression in neurons, we
used embryos carrying elavGAL4 and two copies of UAS-Nkx6
and again focused on ISNb. 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
(Fig. 5). In support of this,
Hb9 expression is wild type in elavGAL4:2XUAS-Nkx6 embryos (data not
shown). However, in this background a significant proportion of ISNb axons
exhibit phenotypes consistent with overgrowth
(Fig. 5). For example, we
observed at least one ISNb branch with a clearly expanded terminal arbor in
28% of hemisegments (n=176; Fig.
5B) compared to 6% in wild type (n=155). We also observed
two phenotypes in Nkx6 overexpression embryos that we never observed
in wild type (n=155). Namely, we observed excessive axonal branching
in ISNb in 14% of hemisegments (n=176;
Fig. 5C). In 4% of
hemisegments, ISNb axons from adjacent segments extend across the segment
boundary and fuse together (n=176;
Fig. 5D). 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.
Nkx6 regulates axon development and gene expression of ventrally projecting RP MNs
The preceding analysis indicates that Nkx6 promotes axon outgrowth
of a subset of MNs. To investigate this possibility in more detail, we
followed the well-characterized axon projections of the RP1,3,4,5 MNs in wild
type and Nkx6 mutant backgrounds. We utilized a lim3-taumyc
transgene (Thor et al., 1999)
to follow RP motor axon projections in wild-type and Nkx6 mutant
embryos (Fig. 6A-D). In wild
type, we are able to detect RP motor axons exiting the CNS in 86% of
hemisegments scored (n=160; arrowheads in
Fig. 6A). In contrast, we could
trace motor axons leaving the CNS in only 39% of hemisegments of Nkx6
mutant embryos (n=210; posterior hemisegments in
Fig. 6C; anterior right
hemisegment in Fig. 6D). In
most mutant hemisegments, the motor axons appear thinner than in wild type
(posterior hemisegments in Fig.
6C). Furthermore, in 61% of Nkx6 mutant hemisegments, RP
motor axons remain within the CNS (n=210; all hemisegments in
Fig. 6B), compared to 14% of
wild type. The morphology of these truncated axons is often aberrant,
suggesting their outgrowth has stalled. For example, we frequently observed
enlarged growth cones with a club-like appearance
(Fig. 6B). Finally, in 10% of
mutant hemisegments, RP motor axons make dramatic guidance errors, often
turning back inappropriately and extending toward the midline (n=210;
anterior left hemisegment in Fig.
6D). 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.
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 as it is expressed by the
RP1,3,4,5 MNs and promotes target recognition by their motor axons
(Fig. 6E)
(Chiba et al., 1995;
Patel et al., 1987
). Notably,
Fas3 expression is strongly reduced in the RP MNs in Nkx6 mutant
embryos relative to wild type (arrowheads in
Fig. 6E,F). Consistent with a
specific role for Nkx6 in regulating Fas3 expression, more lateral
neurons that express Fas3 (arrows, Fig.
6E,F) but not Nkx6 (data not shown) exhibit wild-type Fas3
expression in Nkx6 mutants (Fig.
6E,F). 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.
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Discussion |
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eve represents an appealing candidate for the unidentified
repressor in this model (Fig.
7). Ectopic Eve expression in RP MNs in hb9 Nkx6 double
mutants may repress Lim3 and Islet. Consistent with this, though we were
unable to unambiguously identify the ectopic Eve neurons in hb9 Nkx6
mutants, many of them are situated close to the midline
(Fig. 3F) 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 (data not shown)
(Landgraf et al., 1999). A
direct test of this model will require resolving the identity of the ectopic
Eve neurons in hb9 Nkx6 mutant embryos.
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? We propose 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 (for a review, see
Skeath, 1999). 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 (Schmid et al.,
1999
). Therefore in Drosophila embryos, gene expression
in NBs does not directly promote neuronal subtype identity.
A possible mechanism that might contribute to neuronal subtype identity in
Drosophila is suggested by the temporal gene cascade in NBs
(Isshiki et al., 2001;
Kambadur et al., 1998
). 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
(Schmid et al., 1999
),
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.
Nkx6 and axonogenesis
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 (Nakada et al.,
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 (Brenman
et al., 2001; Grueber et al.,
2003
; Moore et al.,
2002
). 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
(Grueber et al., 2003
).
Interestingly, we find that Nkx6 protein levels vary dramatically and
reproducibly between CNS neurons (Fig.
1). 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.
Our 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 we observe 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 et al.
show that hindbrain visceral MNs in Nkx6.1 mutant mice display
aberrant axon guidance and ectopically express the Ret and Unc5h3 receptors
(Müller et al., 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.
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ACKNOWLEDGMENTS |
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We dedicate this paper to the memory of Nancy C. Tarczy.
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/21/5233/DC1
* Present address: Department of Neurosciences, Case Western Reserve
University School of Medicine, 10900 Euclid Avenue, Cleveland, Ohio 44106,
USA
These authors contributed equally to this work
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