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First published online 21 March 2007
doi: 10.1242/dev.02826
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Institute of Neuroscience, 1254 University of Oregon, Eugene OR 97403, USA.
Author for correspondence (e-mail:
eisen{at}uoneuro.uoregon.edu)
Accepted 31 January 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTS TwoDISCUSSIONREFERENCES |
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Key words: Zebrafish, Motoneuron, Islet1, Nkx6, Interneuron
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS TwoDISCUSSIONREFERENCES |
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;
Shirasaki and Pfaff, 2002).
Specification of motoneuron subtypes has been well-studied in mouse and chick
embryos (Jurata et al., 2000;
Pfaff and Kintner, 1998;
Tsalik et al., 2003); however,
nothing is currently known about the molecular mechanisms that establish very
fine-grained motoneuron patterning, such as the segmentally reiterated,
individually identified primary motoneurons (PMNs) of the zebrafish spinal
cord.
We focus our attention on two PMN subtypes, CaP and MiP, that are present
in a segmentally repeated, alternating pattern
(Lewis and Eisen, 2003). CaPs
project axons that innervate ventral muscle and express the LIM homeobox gene
islet2, whereas MiPs project axons that innervate dorsal muscle and
express the LIM homeobox gene islet1
(Lewis and Eisen, 2003). The
expression of islet genes is dynamic in these cells
(Fig. 1A); both CaP and MiP
express islet1 around the time they exit the cell cycle
(Appel et al., 1995;
Inoue et al., 1994;
Korzh et al., 1993;
Tokumoto et al., 1995). MiPs
then transiently downregulate islet1 expression and reinitiate it
prior to axogenesis (Appel et al.,
1995). Thus, MiPs express islet1 in two distinct phases -
an early phase and a late phase. In contrast to MiPs, CaPs initiate expression
of islet2 while they still express islet1, and then
downregulate expression of islet1
(Appel et al., 1995;
Inoue et al., 1994;
Korzh et al., 1993;
Tokumoto et al., 1995). Thus,
CaPs have an early phase of islet1 expression and a later phase of
islet2 expression. The end result of these dynamic changes in islet
gene expression is that by the time of axon extension, MiPs express
exclusively islet1 and CaPs express exclusively islet2.
Transplantation of single CaPs and MiPs revealed that their subtypes are
initially labile and responsive to environmental signals, but become committed
shortly before axogenesis (Eisen,
1991), around the time that the alternating pattern of
islet2 and islet1 expression is established
(Appel et al., 1995). Thus, it
was surprising to learn that either Islet1 or Islet2 protein is sufficient to
specify both CaP and MiP subtypes, suggesting that the differences between
these PMNs might be regulated by factors upstream of the islet genes
(Hutchinson and Eisen, 2006).
Homeodomain transcription factors expressed in the motoneuron progenitor (pMN)
domain, such as Nkx6.1 (Cheesman et al.,
2004), are good candidates for performing this function.
The Nkx6 transcription factor family is important in formation of mouse
spinal motoneurons. One family member, Nkx6.1, is expressed in the
spinal cord pMN domain and is required for formation of a proportion of all
spinal motoneuron subtypes (Sander et al.,
2000). Another family member, Nkx6.2, is expressed dorsal
to the pMN domain and is negatively regulated by Nkx6.1. In the absence of
Nkx6.1 function, Nkx6.2 expression spreads ventrally into the pMN
domain and Nkx6.2 partially substitutes for Nkx6.1 during motoneuron formation
(Vallstedt et al., 2001). Mice
deficient in both Nkx6.1 and Nkx6.2 lose nearly all their spinal motoneurons
(Vallstedt et al., 2001). As
in mouse, zebrafish Nkx6.1 is expressed in the pMN domain and is also
important for formation of some zebrafish motoneurons
(Cheesman et al., 2004).
Nkx6.1-deficient zebrafish lack secondary motoneurons (SMNs), a type of spinal
motoneuron that develops later than PMNs
(Myers, 1985), but have normal
PMNs, raising the possibility that additional Nkx6 proteins might regulate
zebrafish PMN formation.
Here, we provide evidence for a novel role of Nkx6 proteins in motoneuron subtype specification. We show that zebrafish have at least three nkx6 genes, two of which are expressed in the pMN domain and transiently in postmitotic PMNs. Nkx6 proteins are not required for PMN formation because early islet1 expression is normal in embryos lacking Nkx6 proteins. In Nkx6-deficient embryos, CaPs have normal islet2 expression and project normal ventral axons out of the spinal cord; however, MiPs fail to initiate the second phase of islet1 expression and do not form their subtype-specific, dorsal peripheral axons, instead adopting a more interneuron-like morphology by projecting an axon within the spinal cord. We suggest that Nkx6 proteins control MiP subtype specification at least in part by regulating the second phase of islet1 expression and that this late expression of islet1 is required to promote the MiP subtype and to suppress interneuron development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS TwoDISCUSSIONREFERENCES |
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)
embryos were reared and staged by hours post-fertilization at 28.5°C
(hpf), and gross morphology (Kimmel et
al., 1995).
Cloning
A search of the zebrafish genome revealed an nkx6.2 gene highly
similar to zebrafish nkx6.1
(Cheesman et al., 2004). A
forward primer to an nkx6.2-specific region
(5'-CGGCTTCAAGGCTCATTC-3') and a reverse homeobox primer
(5'-CCATTTAGTTCTTCTGTTCTG-3') isolated a full-length cDNA clone
from a 14- to 19-hpf library (Appel and
Eisen, 1998). These primers did not amplify control
nkx6.1 cDNA. The isolated clone contains an open reading frame and
UTR sequences. The DNA sequence predicts a 279 amino acid protein containing
both a homeodomain and an NK decapeptide, which defines the nkx gene family.
The GenBank accession number for the complete zebrafish nkx6.2 mRNA
is DQ416765. We isolated a fragment of nkx6.3 from first-strand cDNA
(forward primer, 5'-AGTCCAACATCTCAGGATCC-3'; reverse primer,
5'-TCCACTCATACCCTCCATC-3') and based on this clone (GenBank
accession DQ415639) and genomic clones, compiled a putative full-length
sequence.
In situ RNA hybridization
Zebrafish RNA in situ hybridization was performed as described previously
(Appel and Eisen, 1998).
nkx6.1, islet1 and islet2 mRNA probes were described
previously (Appel et al., 1995;
Cheesman et al., 2004), as were
pax2a (Thaeron et al.,
2000), evx1 (Thaeron
et al., 2000), eng1b
(Higashijima et al., 2004) and
chx10 (Kimura et al.,
2006). The nkx6.2 mRNA probe was made from full-length
nkx6.2 DNA. nkx6.3 expression was determined using a probe
against a portion of the nkx6.3 gene that excluded the homeodomain.
In the nkx6.3 anti-GFP double-label experiment, the RNA in situ
hybridization was performed first, followed by antibody detection.
Immunohistochemistry
The following primary antibodies (Abs) were used: polyclonal rabbit
anti-Nkx6.1 (1:1200; gift of O. Madsen, Hagedorn Research Institute, Gentofte,
Denmark), monoclonal mouse anti-Islet [1:200; antibody recognizes both Islet1
and Islet2 proteins (Korzh et al.,
1993); 39.4D5, Developmental Studies Hybridoma Bank], monoclonal
mouse zn1 (Trevarrow et al.,
1990), monoclonal mouse znp1
(Trevarrow et al., 1990),
monoclonal anti-GFP (1:200; Clonetech, JL-8,), and polyclonal rabbit anti-GABA
(1:1000; Sigma). Secondary antibodies from Molecular Probes were used: goat
anti-mouse Alexa-488 (1:1000) and goat anti-rabbit Alexa-568 (1:1000). One
secondary antibody from Jackson Labs was also used: goat anti-rabbit Cy5
(1:200). Embryos were fixed for 3.5-4 hours in 4% paraformaldehyde (PFA) and
1xFix Buffer (Westerfield, 2000) at 4°C, blocked in 1xPBS/5%
NGS/4 mg/ml BSA/0.5% Triton X-100 for 1 hour at room temperature, and
incubated in primary antibody (diluted in blocking solution) overnight at
4°C. Embryos were then washed at room temperature for 1.5 hours in PBS
containing 0.1% Tween 20, incubated in secondary antibody (diluted in blocking
solution) for 4 hours at room temperature, and then washed for 1.5 hours at
room temperature in PBS containing 0.1% Tween 20.
Morpholino injections
The Nkx6.2 conceptual amino acid translation revealed two potential start
methionines; we do not know if one or both of them are functional start sites.
As they are close together, GeneTools (Corvallis, Oregon) designed a
translation-blocking MO just upstream of the first methionine
(5'-GGTGCGCCGGAGCCACAGGACAAAC-3') on the premise that it would
interfere with initiation of translation at either start site. We also
utilized a splice-blocking MO (5'-CGCGCAAAACTCACCCGCACAGGGA-3')
that began at position 386 of the nkx6.2 open reading frame and ended
in the first intron, blocking the splice-donor site of exon 1. Both
nkx6.2 MOs produced similar phenotypes. Several nanoliters of 5 mg/ml
nkx6.2 MO, diluted in 0.2 M KCl, were injected with Phenol Red into
the yolk cell of two-cell-stage embryos; for double MO injections, we added
2.5 mg/ml nkx6.1 MO (see Cheesman
et al., 2004). All our MO injections worked efficiently, with
84.3% (27/32) of nkx6.1 MO-injected, 90.9% (30/33) of nkx6.2
MO-injected, and 76.1% (35/46) of nkx6 double MO-injected embryos
exhibiting a MiP axon phenotype.
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Motoneuron observation and quantification
All observations of CaP and MiP motoneurons were made in the spinal cord
adjacent to somites 8-12. To quantify MiP axons, we counted the number of MiP
axons in 28-hpf control, nkx6 single and double MO-injected embryos,
as well as nkx6 MOs and islet1 RNA co-injected embryos
labeled with zn1 and znp1 Abs. Axons were scored as MiPs if they projected
posterior and dorsal to the zn1-labeled CaP soma. The percentage of MiP axons
remaining in experimental embryos was calculated relative to controls. The
percentage of segments with cells labeled with islet1 RNA in the MiP
position was calculated from cells counted in segments 8-12 of 18-hpf control,
nkx6.1, nkx6.2 and nkx6 double MO-injected embryos. For
experiments in which individual MiPs were dye-labeled, we recognized these
cells by their position and used protocols described by Eisen et al.
(Eisen et al., 1989).
GABA-positive interneurons were counted as described by Hutchinson and Eisen
(Hutchinson and Eisen, 2006).
The number of cells labeled with chx10 or eng1b riboprobes
was counted in segments 1-11 on each side of 24-hpf control and nkx6
double MO-injected embryos. The average number of positive cells per segment
in experimental embryos was compared with that in control embryos.
3D analysis of confocal images
Velocity software was used to generate 3D images of individually labeled
MiPs. A velocity classifier based on intensity was used to generate a
threshold for red and green images of Nkx6 and Islet Abs, followed by Velocity
image arithmetic to identify co-labeled cells. The co-labeled cells were then
pseudo-colored blue.
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RESULTS Two |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS TwoDISCUSSIONREFERENCES |
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) and
showing that zebrafish Nkx6.1 is unnecessary for PMN formation
(Cheesman et al., 2004) raised
the possibility of additional nkx6 genes in zebrafish. We isolated
zebrafish nkx6.2 and nkx6.3 based on publicly available
genomic DNA sequences. Phylogenetic analysis indicated that zebrafish
nkx6 genes are most closely related to other vertebrate nkx6
genes, distinct from nkx2 family members (see Fig. S1A in the
supplementary material). Alignment of the three zebrafish Nkx6 predicted
proteins revealed that Nkx6.1 and Nkx6.2 are more similar to each other than
to Nkx6.3 (see Fig. S1B in the supplementary material). Similar to mouse
Nkx6.3 (Alanentalo et al.,
2006), zebrafish nkx6.3 expression is restricted to a
group of cells in the hindbrain and is not found in the spinal cord (see Fig.
S1C in the supplementary material), and thus it cannot be involved in PMN
development.
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). The
number of nkx6.1-expressing cell rows dorsal to the floor plate
(Cheesman et al., 2004) is
approximately the same as the number of olig2-expressing cell rows
dorsal to the floor plate (see Park et
al., 2002). Thus, like olig2, nkx6.1 is expressed in the
pMN domain (Cheesman et al.,
2004). However, it is also expressed in additional cells ventral
to the pMN domain, and might also be in some cells dorsal to the pMN domain.
At 10 hpf, just before PMN progenitors begin leaving the cell cycle,
nkx6.1 was expressed in more pMN domain cells than nkx6.2
(Fig. 1B,C). At 14 hpf,
expression of nkx6.1 and nkx6.2 were almost
indistinguishable and cross-sections revealed that both genes were confined to
neurectoderm (data not shown). At 18 hpf, nkx6.1 and nkx6.2
expression was no longer apparent in many of the ventrally located cells
(Fig. 1D,E). We previously
showed that the ventral cells lacking nkx6.1 at this stage are PMNs
(Cheesman et al., 2004),
suggesting that expression of both nkx6 genes is extinguished from
PMNs by 17-19 hpf, the time when they undergo axogenesis. Both nkx6.1
and nkx6.2 continued to be expressed in the ventral spinal cord
through at least 24 hpf (see Fig. S2 in the supplementary material).
Nkx6 proteins are differentially expressed in CaPs and MiPs, as shown by
our double-label experiments with an Islet monoclonal antibody that recognizes
both Islet1 and Islet2 proteins (Korzh et
al., 1993), and an Nkx6 polyclonal antibody that recognizes both
Nkx6.1 (Cheesman et al., 2004)
and Nkx6.2 (see Fig. S2 in the supplementary material). At 14 hpf, both CaPs
and MiPs expressed Islet and Nkx6 (Fig.
1F). By 16 hpf, CaPs had extinguished expression of Nkx6 and
expressed only Islet (Fig.
1G-I). By contrast, MiPs downregulated expression of Islet1
protein, similar to their downregulation of islet1 mRNA
(Appel et al., 1995), but
continued to express Nkx6 (Fig.
1G). By 17 hpf, MiPs initiated a second phase of Islet1 protein
expression and continued to express Nkx6
(Fig. 1H). MiPs expressed Nkx6
until 18 hpf, after which time it was downregulated
(Fig. 1I). Nkx6 proteins are
expressed in PMN progenitors (Cheesman et
al., 2004), thus these proteins are present at the right time to
be involved in PMN formation. The differential expression of Nkx6 proteins in
postmitotic MiPs and CaPs suggests that Nkx6 might also participate in
specification of MiP and CaP subtypes.
Nkx6 proteins are not required for primary motoneuron formation
We previously showed that Nkx6.1 is unnecessary for PMN formation
(Cheesman et al., 2004). To
ascertain whether PMNs form in the absence of Nkx6.2, or in the absence of
both Nkx6.1 and Nkx6.2, we injected embryos with morpholino antisense
oligonucleotides (MOs). Early expression of islet1 was normal in the
absence of Nkx6.1, Nkx6.2, or both Nkx6 proteins
(Fig. 2A-D), suggesting that
PMNs formed normally. Thus, although Nkx6 proteins are expressed in the pMN
domain, they appear unnecessary for PMN formation.
The requirement for Nkx6 proteins distinguishes CaP and MiP subtypes
Because CaPs and MiPs continue to express Nkx6 proteins after subtype
commitment, we asked whether Nkx6 proteins have a role in subtype
specification. In the absence of Nkx6.1, Nkx6.2, or both Nkx6 proteins, CaPs
expressed islet2 normally (Fig.
2E-H) and developed normal, ventrally projecting axons
(Fig. 3A-D). Thus, Nkx6
proteins appear unnecessary for CaP subtype specification or axon
pathfinding.
In contrast to CaPs, MiPs were severely affected by the lack of Nkx6 proteins. In the absence of either Nkx6.1 or Nkx6.2, some MiPs failed to initiate the late phase of islet1 expression (Fig. 2I-K). In the absence of both Nkx6 proteins, very few MiPs expressed islet1 at 18 hpf (Fig. 2I,L). In single nkx6 MO-injected embryos, many MiPs had dorsal axons that were truncated or excessively branched and some MiPs failed to form dorsal axons (Fig. 3A-C). MiP axons were present in only 61% of segments in nkx6.1 MO-injected embryos, as compared with 87% of segments in nkx6.2 MO-injected embryos and 98% of segments in control embryos (n=83 segments in nine nkx6.1 MO-injected embryos; n=168 in 17 nkx6.2 MO-injected embryos; n=100 in ten control embryos). MiP axons were most severely affected in embryos injected with both nkx6.1 and nkx6.2 MOs, as indicated by the presence of MiP dorsal axons in only 40% of segments (n=240 in 24 nkx6 MO-injected embryos). The MiP axons that remained in nkx6 MO-injected embryos were severely truncated (Fig. 3D). Thus, Nkx6 proteins are required for proper MiP subtype specification and axon pathfinding.
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MiPs become more interneuron-like in the absence of Nkx6 proteins
The failure to maintain islet1 expression and the absence of
dorsal axons in MiPs lacking Nkx6 proteins led us to consider whether MiPs
developed as interneurons in the absence of late islet1 expression,
as they do in the absence of early islet1 expression
(Hutchinson and Eisen, 2006).
We labeled individual cells in the MiP position in nkx6 double
MO-injected embryos by intracellular dye iontophoresis
(Eisen et al., 1989). As a
negative control, we labeled VeLD interneurons, which are located adjacent to
MiPs (Eisen, 1991), and found
that VeLDs are normal in nkx6 double MO-injected embryos (12 VeLDs in
11 embryos; data not shown). Dye-labeling of MiPs in nkx6 double
MO-injected embryos revealed that these cells had a range of phenotypes
(Fig. 4B-F). MiPs normally
extend a short ventral axon out of the spinal cord before they form their
subtype-specific dorsal axon; the ventral axon is later retracted
(Eisen et al., 1989). In the
absence of Nkx6 proteins, many MiPs projected a normal ventral axon
(Fig. 4C,D), but often failed
to retract it. Some MiPs had a dorsal axon, but often it was excessively
branched (Fig. 4B) or truncated
(Fig. 4C). Surprisingly, many
MiPs initiated motoneuron development by projecting a normal ventral axon, but
then projected an interneuron-like axon within the spinal cord instead of a
dorsal motor axon (Fig. 4D).
Finally, some MiPs did not project either ventral or dorsal motor axons, but
only extended an interneuron-like axon within the spinal cord
(Fig. 4F). However, this
interneuron-like axon was excessively branched compared with axons of any of
the previously described types of ventral spinal interneurons
(Lewis and Eisen, 2003). As an
additional control, we antibody-stained embryos in which we had individually
dye-labeled MiPs to verify that dye-labeled MiPs lacking dorsal axons
correlated with segments lacking dorsal MiP axons as revealed by zn1 and znp1
Ab labeling (Fig. 4G). The
results showed that in the absence of Nkx6 proteins, MiPs formed but they
failed to extend their normal dorsal axon and instead adopted a more
interneuron-like morphology, often developing a `hybrid' phenotype in which
they displayed morphological characteristics of both motoneurons and
interneurons.
|
), but the number of GABA-positive cells did not increase in
nkx6 double MO-injected embryos
(Fig. 5A,B,G), and labeled MiPs
were not GABA positive (Fig.
4G). Thus, we next examined the expression of several
transcription factors that are expressed by interneurons in specific
dorsoventral spinal cord positions. pax2a and evx1
(Thaeron et al., 2000) are
both expressed in interneurons in the dorsal and midregion of the spinal cord;
the evx1 expression domain is just ventral to the pax2a
expression domain. Expression of both pax2a and evx1 were
was in nkx6 double MO-injected embryos (data not shown). By contrast,
chx10 and eng1b, which are both expressed more ventrally
than pax2a and evx1, had altered expression patterns in
nkx6 double MO-injected embryos. Expression of chx10, which
is probably in CiD interneurons (Kimura et
al., 2006), was downregulated in embryos lacking Nkx6
(Fig. 5C,D,G), whereas
expression of eng1b, which is specifically in CiA interneurons
(Higashijima et al., 2004), was
expanded in nkx6 double MO-injected embryos
(Fig. 5E-G). These results are
similar to the expansion of En1 (an eng1b ortholog) and
reduction of Chx10 in mice lacking Nkx6.1 and Nkx6.2
(Vallstedt et al., 2001).
Despite the changes in eng1b and chx10 expression,
dye-labeled MiPs in nkx6 double MO-injected embryos did not express
either of these genes at 28 hpf (data not shown). Therefore, until additional
markers of specific types of interneurons become available, we will remain
unable to ascribe a specific type of interneuron identity to MiPs that develop
in the absence of Nkx6 proteins. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS TwoDISCUSSIONREFERENCES |
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). It
is possible that in zebrafish there are additional nkx6.1 or
nkx6.2 orthologs that we have not discovered. This seems unlikely as
the Nkx6.1 antibody we used (Cheesman et
al., 2004) recognizes both Nkx6.1 and Nkx6.2 proteins (see Fig. S2
in the supplementary material); however, we cannot eliminate the possibility
of additional nkx6 orthologs until the complete sequence of the
zebrafish genome is available. The few spinal motoneurons that form in Nkx6
double-mutant mice appear to develop early, similar to zebrafish PMNs. Thus,
it would be interesting to learn whether early-born motoneurons in mouse have
other features that resemble zebrafish PMNs and distinguish them from
later-developing motoneurons.
Second, we show that the MiP subtype requires Nkx6 proteins, whereas they
appear dispensable for the CaP subtype. In mouse, Nkx6 proteins are required
for formation of most motoneurons
(Vallstedt et al., 2001) (but
see above), but differential function of these proteins in distinct spinal
motoneuron subtypes has not been reported. It will be important in future
studies to identify the factors that regulate CaP subtype specification, as
well as to learn whether Nkx6 proteins have any role in the specification of
mammalian motoneuron subtypes.
Finally, we show that Nkx6 proteins prevent MiPs from developing an
interneuron-like axon by regulating late islet1 expression. We have
previously shown that the early phase of Islet1 expression promotes PMN
formation and inhibits interneuron development
(Hutchinson and Eisen, 2006).
Here, we suggest that the second phase of Islet1 expression promotes the MiP
subtype and also inhibits interneuron axon development. However, in the
absence of only the late phase of Islet1 expression, MiPs often adopt a hybrid
motoneuron/interneuron identity, rather than developing as interneurons, as
they do in the absence of both phases of Islet1 expression.
The ability of zebrafish Nkx6 proteins to prevent MiPs from developing an
interneuron-like axon is reminiscent of the ability of mouse Nkx6 proteins to
inhibit pMN-domain cells from expressing interneuron-specific transcription
factors (Vallstedt et al.,
2001). Interestingly, Vallestedt and colleagues reported that in
the absence of Nkx6 proteins, motoneurons transiently express Evx1,
although whether these cells developed interneuron-like axons was not
reported. We did not observe ectopic evx1 expression; however, it is
possible that evx1 might be expressed early and transiently in MiPs
in the absence of Nkx6 proteins. Mouse Nkx6 proteins apparently act within
motoneuron progenitors. By contrast, our data raises the possibility that in
MiPs, Nkx6 proteins function later, perhaps after the cell has become
postmitotic. It will be important to address the timing of Nkx6 function
further in future studies.
It is interesting that in the absence of Nkx6 proteins, MiPs become more
interneuron-like, rather than becoming more like CaP. MiPs can develop as CaPs
when they are transplanted to the CaP spinal cord position several hours
before axogenesis (Appel et al.,
1995; Eisen, 1991).
However, when MiPs are in their normal spinal cord position, they are unlikely
to transform into another PMN subtype because specification of MiP and CaP
subtypes requires positional signals derived from the overlying somites
(Lewis and Eisen, 2004). Our
data support a model in which interneuron formation is continuously suppressed
during motoneuron development. It will be important to determine whether
interneuron specification similarly requires continuous suppression of
motoneuron development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/9/1671/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS TwoDISCUSSIONREFERENCES |
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