1 Institute of Neuroscience, 1254 University of Oregon, Eugene OR, 97403-1254,
USA
2 Institute of Molecular Biology 1229 University of Oregon, Eugene OR,
97403-1229, USA
3 Howard Hughes Medical Institute, 1254 University of Oregon, Eugene OR,
97403-1254, USA
Author for correspondence (e-mail:
eisen{at}uoneuro.uoregon.edu)
Accepted 12 August 2004
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SUMMARY |
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Key words: nkx6.1, Danio rerio, Drosophila melanogaster, Hedgehog, Primary motoneurons, Secondary motoneurons, Interneurons, Eve, HB9, Islet
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Introduction |
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Although these genes are expressed in strikingly similar spatial patterns
in flies and mice, not all of them are expressed at corresponding
developmental stages. All three fly genes are expressed early during CNS
development (Chu et al., 1998;
Isshiki et al., 1997
;
McDonald et al., 1998
;
Weiss et al., 1998
), whereas
only Msx genes are expressed at a corresponding stage in the mouse
(Satokata and Maas, 1994
;
Wang et al., 1996
). Thus, the
hypothesis that similar mechanisms underlie dorsoventral patterning of
arthropod and vertebrate neurectoderm predicts that vertebrates should have as
yet uncharacterized, early-expressed paralogs of the fly genes.
Nkx genes are expressed in ventral spinal cord in mouse, chick and
zebrafish. Nkx2.2, an ortholog of fly vnd, is required to
establish a ventral interneuron precursor domain in the mouse
(Briscoe et al., 1999).
However, zebrafish nkx2.2 is expressed relatively late in ventral
spinal cord (Barth and Wilson,
1995
), precluding involvement in early neural patterning.
Nkx6.1, a family member that might fulfill an earlier patterning role
has been described in the mouse and chick
(Qiu et al., 1998
). This gene
is expressed early in medial neural plate and later in ventral spinal cord.
Nkx6.1 is sufficient for motoneuron formation in the chick and required for
formation of a subset of motoneurons and interneurons in the mouse
(Briscoe et al., 2000
;
Sander et al., 2000
). However,
a dramatic reduction in spinal motoneurons is only seen in mice lacking both
Nkx6.1 and a related gene, Nkx6.2, suggesting their
functions overlap (Vallstedt et al.,
2001
). Flies also have an Nkx6 gene that is expressed early in a
subset of ventral neuroblasts and later in CNS neurons
(Uhler et al., 2002
). Thus,
Nkx6 genes in flies and vertebrates represent another pair of orthologs
expressed in similar CNS domains.
To determine whether Nkx6 genes play similar roles in specifying ventral
CNS identity in vertebrates and arthropods, we investigated Nkx6
function in zebrafish and flies. We cloned and characterized a zebrafish
nkx6.1 gene that is expressed initially in ventral CNS and later in
motoneurons and some interneurons. Like other anamniote vertebrates, zebrafish
have two distinct types of spinal motoneurons. Primary motoneurons (PMNs) are
individually identifiable, born early, and have axons that pioneer motor
nerves (Eisen et al., 1986).
Secondary motoneurons (SMNs) are more numerous, born later
(Myers, 1985
), and have axons
that follow primary motor axons (Pike et
al., 1992
). The ventral spinal cord domain that generates PMNs and
SMNs also generates oligodendrocytes and at least three types of interneurons;
Komer-Agduhr (KA), Ventral Longitudinal Descending (VeLD) and Circumferential
Descending (CiD), each of which can be identified by soma position and axonal
trajectory (Appel et al., 2001
;
Bernhart et al., 1992
;
Park et al., 2002
) (H. C. Park
and B. Appel, personal communication).
Here we show that ectopic expression of Nkx6 genes in zebrafish or flies results in embryos with supernumerary motoneurons, suggesting that at least one function of Nkx6 genes has been preserved over the last 800 million years. Surprisingly, knocking down Nkx6.1 protein in zebrafish reduces the number of SMNs, but not PMNs, raising the possibility of additional nkx6 genes. Nkx6.1 knockdown also increases the number of VeLD interneurons, suggesting that Nkx6.1 protein regulates a decision between motoneuron and interneuron fate, consistent with our finding that there are fewer VeLDs and supernumerary motoneurons in embryos ectopically expressing Nkx6.1. Fly embryos ectopically expressing either fish or fly Nkx6 show a similar phenotype.
The apparent conservation of Nkx6 expression and function in flies and fish suggested these genes might be similarly regulated. As in other vertebrates, Hedgehog (Hh) signaling is necessary for expression of zebrafish nkx6.1. However, expression of fly Nkx6 is unaffected by lack of Hh. These data suggest that although the signals establishing Nkx6 expression have diverged, Nkx6 proteins function as an ancient patterning mechanism to establish motoneurons within the CNS.
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Materials and methods |
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Drosophila melanogaster embryos were gathered from staged collections. Adults of the following genotypes were induced to lay embryos on a grape agar pad coated with yeast paste; yw, sca>Nkx6 (fly gene), sca>nkx6.1 (zebrafish gene), eg>Nkx6 (fly gene), w1118;hhts2 e/TM3 ftz lacZ, hh6n16e/TM3 ftz lacZ.
Zebrafish nkx6.1 cDNA isolation
The following degenerate primers amplified an approximately 730 base
fragment from 10 hpf cDNA (Ambion RETROScript Kit), forward
5'-TGCACTCCATGGCCGARATGAARAC-3', reverse
5'-CGCCGGTTCTGGAACCANACYTT-3'. We designed specific primers and
screened a gastrula stage cDNA library
(Faucourt et al., 2001) to
isolate a full-length clone; forward 5'-GCCTACCCGTTATCTTCCACT-3',
reverse 5'-GACTTGACTCTCTGTCGTTCCT-3'. Zebrafish nkx6.1
GenBank Accession number is AY437556.
The Fly EST database
(http://flybase.bio.indiana.edu)
contains four identical clones with high similarity to the zebrafish
nkx6.1 homeodomain and NK domain, indicating the fly genome probably
contains a single Nkx6 gene, designated conceptual gene
CG13475, also known as Nk6
(Uhler et al., 2002).
Zebrafish nkx6.1 and fly Nkx6 (ResGen EST clone RE66661)
ORFs trimmed of UTRs were inserted into the pCS2+ MT expression
vector (Rupp et al., 1994) for
use in overexpression experiments.
Phylogenetic analysis
MacVector software, utilizing the Clustal W algorithm, created a maximum
likelihood tree. Sequences included in the tree were gathered from
GenBank.
In situ hybridization
Zebrafish RNA in situ hybridization was performed as described by Appel and
Eisen (Appel and Eisen, 1998).
RNA probes include islet1 and islet2
(Appel et al., 1995
). RNA in
situ hybridization on fly embryos was performed as described by Tautz and
Pfeifle (Tautz and Pfeifle,
1989
).
Immunohistochemistry
Zebrafish
The following antibodies were used: monoclonal mouse anti-Islet
(Korzh et al., 1993)
recognizes the Islet1 and Islet2 proteins (1:200; 39.4D5 Developmental Studies
Hybridoma Bank), polyclonal rabbit anti-Nkx6.1 (1:1200; gift of O. Madsen),
anti-Neurolin (1:4000) (Trevarrow et al.,
1990
) (also known as zn5 and DMGRASP;
www.zfin.org),
polyclonal anti-GABA (1:1000, Sigma), zn1 monoclonal (1:200)
(Trevarrow et al., 1990
) and
znp1 monoclonal (1:1000) (Trevarrow et
al., 1990
). Embryos were processed as described by Appel et al.
(Appel et al., 2001
).
Flies
The following antibodies were used: mouse anti-Engrailed (1:5)
(Patel et al., 1989)
(Developmental Studies Hybridoma Bank), mouse anti-Eve (1:20; N. Patel), mouse
anti-Eagle (1:500; M. Freeman and C. Doe), rabbit anti-Vnd (1:20)
(McDonald et al., 1998
),
rabbit anti-Odd (1:100) (Spana and Doe,
1995
), rat anti-Ind (1:250)
(Weiss et al., 1998
), mouse
anti-FasII (1:100; C. Goodman), rat anti-Islet, guinea pig anti-HB9 (both
1:500) (Broihier and Skeath,
2002
), rat anti-HB9 (1:500)
(Odden et al., 2002
), rabbit
anti-pMAD (1:300) (Marques et al.,
2002
). Embryos were processed as described by Odden et al.
(Odden et al., 2002
).
Microscopy
Images of zebrafish and fly embryos were captured on a Zeiss Axioplan
equipped with a digital camera, or a Bio-Rad Radiance confocal microscope.
Adobe Photoshop was used to adjust brightness and contrast of images.
RNA and morpholino injections
shh mRNA (shh p64-T)
(Krauss et al., 1993),
nkx6.1 mRNA (pCS2-MT), and Nkx6 mRNA (pCS2-MT) were
transcribed using the mMessage mMachine kit (Ambion) according to
instructions. Two-cell stage embryos were injected with several nanoliters of
either 0.1 mg/ml, 2 mg/ml and 2 mg/ml of shh, nkx6.1 and
Nkx6 RNA, respectively. Numbers of PMNs were counted adjacent to
somites 5-17 and subjected to a Student's t-test to determine
significance.
To create zebrafish embryos with severely reduced Hh signaling,
tiggywinkle hedgehog (twhh) and echidna hedgehog
(ehh) mopholino antisense oligonucleotides (MOs) were injected as in
Lewis and Eisen (Lewis and Eisen,
2001). An nkx6.1 translation-blocking MO beginning at
position 60 in the 5' UTR,
(5'-CGCAAGAAGAAGGACAGTGACCCG-3') was designed by Gene Tools
(Corvallis, Oregon). Several nanoliters of 2.5 mg/ml MO were injected as
described by Lewis and Eisen (Lewis and
Eisen, 2001
). MO-injected embryos generally looked healthy but had
little or no Nkx6.1 protein in the spinal cord at all stages assayed. Embryos
injected with a 5-base mispair MO (5'-CGgAAGAAcAAcGACAcTGAgCCG-3')
had a wild-type pattern of Nkx6.1 protein in the CNS.
Single cell labeling
Individual PMNs and VeLD interneurons were labeled using the methods of
Eisen et al. (Eisen et al.,
1989) and detected with an anti-fluorescein antibody (1:1000,
Boehringer Mannheim).
BrdU and TUNEL labeling
BrdU labeling was performed as described in Appel et al.
(Appel et al., 2001). TUNEL
labeling was performed as described by Reyes et al.
(Reyes et al., 2004
).
Generation of Fly UAS lines
A portion of fly Nkx6 (EST clone RE66661, Research Genetics) and
zebrafish nkx6.1 sequences trimmed of UTRs were subcloned into the
pUAS vector (Brand and Perrimon,
1993). Independent injection of these plasmids produced founding
lines carrying the red eye marker (mini-white) that were outcrossed to
yw flies. Transgenic progeny from this cross were crossed to the
balancer lines w;Gla/Cyo and w;TM3/TM6 to determine which
chromosome carried the inserted transgene, and homozygous stocks were
established.
RNAi on fly embryos
Two non-overlapping 500 base fragments of the Nkx6 coding region,
excluding the homeodomain, were amplified by PCR for use as templates. RNA was
injected as described by Sullivan et al.
(Sullivan et al., 1999
).
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Results |
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Zebrafish nkx6.1 is expressed in ventral spinal cord
To test our prediction that zebrafish nkx6.1 would be expressed in
ventral CNS, we performed RNA in situ hybridization. Transcripts were first
detected at the onset of gastrulation in the embryonic shield epiblast
(Fig. 3A). Near the end of
gastrulation, nkx6.1 was expressed in medial neurectoderm in two
wide, diffuse stripes (Fig.
3B). By the 3-4 somite stage, nkx6.1 was confined to a
tight stripe in medial neural keel, extending from the midbrain through the
posterior neural plate. This pattern was maintained throughout somitogenesis
(Fig. 3D); Nkx6.1 expression
persisted in ventral spinal cord until at least 48 hpf (data not shown).
nkx6.1 was detected in ventral hindbrain caudal to the
midbrain-hindbrain boundary and in pancreas at later stages
(Fig. 3C). To test for control
at the level of translation, we labeled zebrafish embryos with a polyclonal
anti-Nkx6.1 antibody designed against the highly conserved C-terminus of rat
Nkx6.1 (Jensen et al., 1996).
Although we cannot rule out the possibility that Nkx6.1 antibody crossreacts
with additional Nkx6 proteins, at all stages examined protein and RNA patterns
appeared indistinguishable (data not shown). Cross-sections of 24 hpf embryos
revealed Nkx6.1 expression in about five longitudinal cell rows in ventral
spinal cord, including both medial and lateral floorplate
(Fig. 4C); 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
(Park et al., 2002
), 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 (Kimmel et
al., 1994
; Myers et al.,
1986
). To determine whether postmitotic motoneurons express
Nkx6.1, we performed antibody double-label experiments. Islet
(Appel et al., 1995
;
Korzh et al., 1993
) and Nkx6.1
proteins are colocalized in PMNs during early somitogenesis (14 hpf,
Fig. 4A); 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
(Fig. 4B). Cross-sections at 48
hpf revealed Nkx6.1-positive SMN nuclei surrounded by Neurolin-positive plasma
membranes (Fashena and Westerfield,
1999
), indicating co-expression in these cells
(Fig. 4D and data not shown).
Thus both PMNs and SMNs express Nkx6.1 at least transiently.
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Fly Nkx6 is expressed in ventral neuroblasts and motoneurons
Nkx6 expression was first detected during early neurogenesis
(stage 9) in the nerve cord midline, and it was weakly expressed in ventral
column neurectoderm of rostral segments
(Fig. 5A). An hour later (early
stage 10), Nkx6 midline and neurectoderm expression was
downregulated. Nkx6 expression was restricted to six ventral column
neuroblasts, rostrally located in each hemisegment
(Fig. 5B). Consistent with
Uhler and colleagues (Uhler et al.,
2002) we detected Nkx6 expression in neuroblasts 2-2,
3-1, 3-2 and 4-2 (Fig. 5D). We
also found Nkx6 expression in neuroblasts 1-1 and 1-2
(Fig. 5E,F). 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 was expressed in a segmentally reiterated pattern
of CNS neurons (Fig. 5C). At
stage 14, many of these Nkx6-positive cells also expressed the postmitotic
motoneuron marker, pMAD (Marques et al.,
2002
) (Fig. 5H)
suggesting that many, perhaps all, motoneurons are initially Nkx6-positive.
However, at later stages Eve-positive motoneurons no longer expressed Nkx6
(Fig. 5G), consistent with the
observation that the CNS contained Nkx6-negative, pMAD-positive motoneurons
(Fig. 5I). 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.
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Hh expression is very different in vertebrate and fly embryos (reviewed by
Eisen, 1998). In vertebrates,
Hh is expressed in axial neurectoderm (floorplate) and underlying axial
mesoderm (notochord). In contrast, in fly neurectoderm Hh is expressed in
segmentally reiterated stripes orthogonal to the CNS midline. These stripes
also express Engrailed (Tabata et al.,
1992
; Mohler and Vani,
1992
), a marker for row 6 and 7 neuroblasts
(Broadus et al., 1995
). Thus,
the Hh expression domain is adjacent to the six Nkx6-positive
neuroblasts, raising the possibility that Hh might regulate Nkx6 expression in
these cells. To test this, we examined Nkx6 expression in Hh mutants, which
lack neuroblasts in rows 2, 5 and 6
(Matsuzaki and Saigo, 1996
),
including Nkx6-positive neuroblast 2-2. The Nkx6 expression
pattern in the remaining five Nkx6-positive neuroblasts was wild type
(data not shown), suggesting that flies utilize a different mechanism from
vertebrates to establish Nkx6 expression. We also assessed whether Hh
was required for formation of motoneurons derived from an Nkx6-positive
neuroblast by examining co-expression of HB9 and pMAD in the RP1,3,4,5
motoneuron progeny of neuroblast 3-1. We found no change in these motoneurons
in homozygous hh mutant embryos (data not shown), suggesting that Hh
signaling is unnecessary for their formation.
Nkx6.1 is sufficient to generate supernumerary motoneurons and suppress interneurons in zebrafish
Because in zebrafish nkx6.1 is expressed in motoneurons and their
progenitors, we tested whether nkx6.1 was sufficient to generate
these cells. All zebrafish 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 (Appel et al., 1998;
Tokumoto et al., 1995). We
injected synthetic nkx6.1 mRNA and assayed for the presence of
isl1-positive or isl2-positive PMNs by RNA in situ
hybridization at 18 hpf. The isl1 probe revealed supernumerary MiPs
and RoPs (data not shown) and the isl2 probe revealed supernumerary
CaPs and VaPs (Fig. 7A,B),
which we confirmed by labeling individual CaPs and VaPs in
nkx6.1-injected embryos with fluorescent dextrans (data not shown).
We also observed large clusters of zn1-positive motoneurons projecting axons
into the periphery (Fig. 7D) as
compared to wild types (Fig.
7C). Many of these supernumerary PMNs were located more dorsally
than native PMNs (Fig. 7A-D),
suggesting that Nkx6.1 converted some dorsal cells to a ventral fate.
Injection of a GATA-2:GFP transgenic line that expresses GFP
predominantly in SMNs (Meng et al.,
1997
) (Fig. 7E)
with synthetic nkx6.1 mRNA revealed supernumerary SMNs at 24 hpf
(Fig. 7F). Because Nkx6.1 is
expressed in VeLD interneurons (Fig.
4E,F), we also tested whether overexpression of Nkx6.1 affected
this cell type. VeLDs are recognized by cell body position and GABA expression
(Bernhardt et al., 1992
).
Embryos ectopically expressing nkx6.1 RNA have fewer GABA-positive
VeLDs than wild types at 24 hpf (Fig.
7G,H), suggesting cells that would normally become VeLDs become
motoneurons instead.
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Nkx6.1 is required for formation of zebrafish secondary motoneurons
To test whether Nkx6.1 is required for motoneuron formation, we injected
embryos with an nkx6.1-specific MO
(Fig. 8A,B and data not shown).
Surprisingly, nkx6.1 MO-injected animals had normal numbers of PMNs,
revealed by isl1 expression at 12 hpf and isl2 expression at
18 hpf (Fig. 8C,D and data not
shown). MO-injected embryos initiated the spontaneous tail reflex around 18
hpf, thus PMNs were functional
(Saint-Amant and Drapeau,
1998). 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 did not swim when touched, suggesting an
absence of SMNs. Consistent with this, at 48 hpf, nkx6.1 MO-injected
animals consistently had fewer SMNs (Fig.
8F). At this stage there are more than 30 SMNs per spinal
hemisegment; these cells are difficult to count because their somata are
closely packed (Fig. 8E). Thus,
we divided MO-injected embryos 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 (Fig. 8F); in a few cases
SMNs were entirely absent. To test whether SMNs were dying in MO-injected
embryos, we performed TUNEL assays 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
(data not shown). We also tested whether decreased proliferation accounted for
the decrease in SMNs in MO-injected embryos. BrdU incorporation showed no
difference between MO-injected and wild-type embryos at 24, 30 and 36 hpf
(data not shown).
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Zebrafish and fly Nkx6 gene function is conserved in CNS patterning
We addressed whether Nkx6 genes have conserved functions by overexpressing
the fly gene in zebrafish and the zebrafish gene in flies. We 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
(Fig. 9B,D) as compared to wild
types (Fig. 9A,C), similar to
the phenotype of embryos overexpressing zebrafish nkx6.1
(Fig. 7); both fish and fly
Nkx6 appear equally potent at generating ectopic PMNs in
zebrafish.
|
We next assayed embryos misexpressing fly or zebrafish Nkx6 genes 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 (Broihier et al., 2002;
Landgraf et al., 1999;
Marques et al., 2002
;
Odden et al., 2002
).
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 (Fig. 10H,J).
There was also occasional duplication of the Eve-positive RP2 motoneuron
(Fig. 10A,B). We conclude 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 (Fig.
10E-G) and one type was slightly decreased, the Eve-positive U
motoneurons (Fig. 10C,D). We
also saw consistent loss of identified interneurons, including the
Eve-positive ELs and Islet-positive EWs
(Fig. 10C-F). 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.
|
To examine potential lineage effects, we expressed fly Nkx6 under the control of Eagle (Eg), which is expressed in neuroblast 7-3 and its progeny, the HB9-positive EW interneurons and GW motoneuron. We found the same number of Eg-positive, HB9-positive cells in controls and embryos overexpressing Nkx6 (2.65±0.69 in 26 control hemisegments and 2.64±0.53 in 42 hemisegments overexpressing Nkx6; P<0.34). However, more than twice as many of these cells expressed pMAD in embryos overexpressing Nkx6 than in controls (0.85±0.83 in 26 control hemisegments; 1.93±0.81 in 42 hemisegments overexpressing Nkx6; P<0.002) 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.
We also tested whether Nkx6 was necessary for fly motoneuron formation by
RNAi. We saw no change in the numbers of HB9 or pMAD-positive cells in embryos
lacking Nkx6, suggesting that it is not required for motoneuron formation
(data not shown), consistent with the phenotype of Nkx6 mutants
(Broihier et al., 2004).
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Discussion |
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Zebrafish Nkx6.1 promotes motoneuron and suppresses interneuron formation
Nkx6.1 is expressed in a zebrafish spinal cord domain that generates
diverse cell types, including PMNs and SMNs, interneurons and oligodendroctyes
(Park et al., 2002) (H. C.
Park and B. Appel, personal communication). We focused on the role of
nkx6.1 in motoneurons and VeLD interneurons. However, a complete
understanding of how this gene functions requires lineage analysis to
ascertain when it is expressed in each cell type generated in this region, and
how these cell types are related to one another. In zebrafish, Nkx6.1 is
required for formation of SMNs, but not PMNs. This is surprising considering
that in mice lacking Nkx6.1 all subtypes of spinal motoneurons are
similarly depleted (Sander et al.,
2000
). Thus, we expected to see fewer PMNs and SMNs in zebrafish.
It would be interesting to learn whether mice lacking Nkx6.1 are missing
specific motoneuron subsets, such as later-generated motoneurons. Nkx6.1 is
one of the few known proteins that differentially affects SMN and PMN
formation in zebrafish, raising the possibility that other, related genes may
participate in PMN formation. A good candidate is Nkx6.2, which is
able to substitute for Nkx6.1 in mice lacking Nkx6.1 function
(Vallstedt et al., 2001
).
Interestingly, mouse Nkx6.2 is not normally expressed in the motoneuron
progenitor domain, but its expression expands into this domain in the absence
of Nkx6.1. It will be important to learn whether zebrafish has an Nkx6.2
homolog, where it is expressed and if it is required for PMN formation.
The decreased number of SMNs in nkx6.1 MO embryos was accompanied
by an increase in VeLD interneurons. Conversely, embryos ectopically
expressing nkx6.1 had fewer VeLDs. The simplest interpretation of
these results is that when Nkx6.1 is present, ventral cells become SMNs and
when it is absent, they become interneurons. However, we cannot rule out the
possibility that VeLDs remain in the absence of Nkx6.1, but no longer express
GABA and thus are not apparent in our assay. This could be resolved by
developing a more specific probe for VeLDs that would allow them to be
observed at a variety of developmental stages in wild-type and mutant embryos.
Despite the need for further study in zebrafish, we favor the idea that Nkx6.1
promotes a motoneuron program and represses the VeLD interneuron program
because such a role would be consistent with the function of Nkx6.1 in mouse
ventral spinal cord (Sander et al.,
2000; Vallstedt et al.,
2001
). It will be interesting to learn whether other interneurons
from the same domain of the zebrafish spinal cord are similarly regulated.
Fly Nkx6 generally promotes motoneuron and suppresses interneuron formation
Overexpression of fly Nkx6 in fly CNS produced a complex phenotype
that might result from fate changes within neuroblast lineages. For example,
cells in the EW interneuron position often expressed pMAD, a definitive
motoneuron marker, suggesting an interneuron-to-motoneuron fate change. There
are many more motoneurons in the lateral cluster than can be simply explained
by EWs becoming motoneurons; thus other sources must also contribute to
formation of supernumerary, HB9-positive, lateral cluster motoneurons.
HB9-positive motoneurons project to ventral muscles via SNb (Broihier et al.,
2002; Odden et al., 2002),
thus, they probably contribute to the thicker SNb motor nerves. Both HB9 and
Islet are required for proper ventral motoneuron projections
(Broihier and Skeath, 2002
;
Odden et al., 2002
;
Thor and Thomas, 1997
),
suggesting Nkx6 interacts with them during this process.
Nkx6 and Eve are expressed in mutually exclusive neuronal subpopulations at
the end of fly gastrulation. All dorsally projecting motoneurons are
Eve-positive (Landgraf et al.,
1999) and thus Nkx6 must be restricted to interneurons or the
ventrally projecting, Islet-positive, HB9-positive motoneurons at this
developmental stage. However, at earlier stages Nkx6 is expressed in some
neuroblasts that generate Eve-positive motoneurons and probably in those
motoneurons themselves. Thus, in flies as in fish, Nkx6 expression is
apparently downregulated in at least some motoneurons. However, Nkx6 must play
a role in the generation of these cells as overexpression causes duplicated
Eve-positive RP2 motoneurons and fewer Eve-positive U motoneurons.
Understanding how expression of Eve and Nkx6 is regulated in these cells
should elucidate when Nkx6 acts during motoneuron formation.
Are Nkx6 genes ancient regulators of motoneuron development?
Several dorsoventral patterning genes are expressed in strikingly similar
patterns in the developing CNS of vertebrates and flies, motivating us to test
whether these genes have conserved functions. Mouse Nkx2.2 and Gsh-1,
orthologs of fly Vnd and Ind, produced no phenotype when overexpressed in fly
embryos, revealing that the vertebrate proteins do not function in the context
of the other species (T.V.O., unpublished) and suggesting functional domains
have become distinct over time. That zebrafish Nkx6.1 and fly Nkx6 have
similar overexpression phenotypes suggests their functions in CNS patterning
have been preserved over 800 million years of evolution. We propose the
bilaterian ancestor utilized Nkx6 proteins both to pattern ventral CNS and to
promote motoneuron and suppress interneuron development. In vertebrates,
Nkx6.1 is ventrally expressed, consistent with the ventral origin of CNS
motoneurons. In flies, Nkx6 is also initially ventrally restricted, although
its expression is not strictly correlated with motoneuron-producing
neuroblasts, which are distributed throughout the CNS dorsoventral axis.
Later, fly Nkx6 is expressed at least transiently in many, perhaps all,
developing motoneurons, thus it must be expressed in some motoneurons whose
neuroblast antecedent was Nkx6-negative. This suggests that expression of Nkx6
is differentially regulated in neuroblasts and their progeny.
How flies generate motoneurons from all CNS dorsoventral levels and
vertebrates from only ventral CNS remains a mystery. Thor and Thomas
(Thor and Thomas, 2002)
proposed that the common ancestor of vertebrates and arthropods had both
dorsally and ventrally projecting motoneurons and that vertebrates lost the
dorsally projecting subset because of constraints imposed by evolution of the
notochord. This is consistent with the observation that the ventrally
projecting motoneurons in both flies and vertebrates express the same
transcription factors, Nkx6, Islet, HB9 and Lim3. The origin of motoneurons
can be addressed by studying nervous system patterning in other protostome and
deuterostome phyla to learn whether ventral restriction of motoneurons is a
basal or derived characteristic.
Transcription factors that control motoneuron formation are regulated by different mechanisms in vertebrates and arthropods
The inputs establishing Nkx6 expression have apparently diverged
between vertebrates and arthropods. In flies there are at least two spatially
and temporally distinct phases of Nkx6 expression, early in ventral
neuroblasts and later in neurons, only some of which are derived from
Nkx6-positive neuroblasts. This has allowed us to ask whether the same
signaling mechanism affects Nkx6 function at different times and in different
cell types. Because Hh is required for Nkx6 expression in vertebrates, we
asked whether Hh also affected Nkx6 in flies. We found that Hh does not affect
Nkx6 expression in fly neuroblasts, showing that it is not involved
in establishing the early pattern. We also found that motoneuronal progeny of
at least one Nkx6-positive neuroblast develop in the absence of Hh signaling,
thus Hh is also probably not involved in establishing the later pattern of
Nkx6 expression. What regulates formation of fly motoneurons is still an
enigma.
In contrast to flies, Hh is required to induce nkx6.1 in all vertebrates tested thus far, including zebrafish. Because motoneurons arise from progenitors in the ventral domain it is unclear whether Nkx6 is regulated differently in progenitors and their progeny. This could be tested by downregulating Hh at later developmental stages.
The ability of ectopic nkx6.1 to induce supernumerary PMNs in
zebrafish raises a conundrum. Hh is both necessary and sufficient for
nkx6.1 expression, however we and others have found that ectopic Hh
is insufficient to induce supernumerary PMNs except in the most rostral spinal
cord (Hammerschmidt et al.,
1996) (S.E.C. and J.S.E., unpublished). How can a downstream gene
be sufficient to generate a particular cell type when the upstream gene that
induces it is insufficient? One possibility is that Hh promotes expression of
both positive and negative regulators of motoneuron formation. Normal levels
of Hh lead to a balance between these negatively acting and positively acting
downstream genes, and PMNs are formed. In contrast, excess Hh tips the balance
in favor of negative regulators, thus preventing formation of supernumerary
PMNs. Hh is known to induce other factors that regulate motoneuron formation
such as olig2, which can only generate PMNs in concert with Hh
(Park et al., 2002
), showing
that additional Hh-dependent factors are required; nkx6.1 is a good
candidate for fulfilling this role.
It is clear that although the same transcription factors regulate formation of ventrally projecting motoneurons in vertebrates and arthropods, these transcription factors are regulated by different mechanisms in these distinct taxa. It will be exciting to learn what regulates the Nkx6 pathway leading to motoneuron formation in flies and whether formation of dorsally projecting and of ventrally projecting motoneurons are regulated by the same mechanisms.
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ACKNOWLEDGMENTS |
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We dedicate this paper to our dear friend and colleague José Campos-Ortega.
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Footnotes |
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