1 Howard Hughes Medical Institute, Department of Biochemistry and Molecular
Biophysics, Center for Neurobiology and Behavior, Columbia University, 701
West 168th Street, New York, NY 10032, USA
2 Mitsubishi Kagaku Institute of Life Sciences, PREST, Japan Science and
Technology, 11 Minami-Ooya, Machida-shi, Tokyo 194 8511, Japan
* Author for correspondence (e-mail: tmj1{at}columbia.edu)
Accepted 23 December 2002
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SUMMARY |
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Key words: Motor neuron, Motor neuron specification, Mnx class homeodomain proteins, Transcriptional repression
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INTRODUCTION |
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One region of the CNS in which some progress has been made in linking
inductive signaling and transcription factor expression to neuronal fate is
the developing spinal cord (Jessell,
2000; Shirasaki and Pfaff,
2002
), where the pathway of motor neuron specification has been
defined in greater detail than for other neuronal classes
(Briscoe and Ericson, 2001
).
The generation of motor neurons depends initially on the graded signaling
activity of Sonic hedgehog (Shh), an inductive factor secreted by the
notochord and floorplate (Chiang et al.,
1996
; Ericson et al.,
1996
; Patten and Placzek,
2000
; Litingtung and Chiang,
2000
). Shh signaling specifies the identity of motor neuron
progenitors by regulating the pattern of expression of a set of homeodomain
(HD) and basic helix-loop-helix (bHLH) transcription factors that fall into
two major groups: a set of class I proteins that are repressed by Shh
signaling; and a set of class II proteins that are activated by Shh
(Briscoe et al., 2000
;
Briscoe and Ericson, 2001
).
These proteins function primarily as transcriptional repressors
(Muhr et al., 2001
), and their
selective cross-regulatory interactions help to establish specific neural
progenitor domains and to sharpen the boundaries between these domains
(Briscoe et al., 1999
;
Briscoe et al., 2000
;
Jessell, 2000
;
Vallstedt et al., 2001
).
Motor neuron progenitors are restricted to a narrow region of the ventral
neural tube that has been termed the pMN domain
(Briscoe et al., 2000;
Jessell, 2000
;
Pierani et al., 2001
). The
cross-regulatory interactions between class I and class II proteins that
establish the pMN domain lead, in turn, to the expression of a distinct set of
downstream transcription factors that include the HD proteins MNR2 and Lim3
(Tsuchida et al., 1994
;
Tanabe et al., 1998
;
Sharma et al., 1998
). MNR2 is
a member of an evolutionarily conserved subgroup of Mnx class HD proteins
(Ferrier et al., 2001
), which
includes the vertebrate HB9 (Hlxb9) (Pfaff
et al., 1996
; Saha et al.,
1997
; Ross et al.,
1998
; Tanabe et al.,
1998
) and Drosophila HB9
(Broihier and Skeath 2002
)
proteins, and homologs in sea urchin and amphioxus
(Bellomonte et al., 1998
;
Ferrier et al., 2001
). The
vertebrate HB9 protein is expressed by postmitotic motor neurons, and genetic
studies in mouse have revealed its role in the consolidation of motor neuron
identity (Arber et al., 1999
;
Thaler et al., 1999
). More
recently, a similar function for Drosophila HB9 has been demonstrated
during Drosophila motor neuron development
(Broihier and and Skeath,
2002
). In the chick, the expression of MNR2 differs from Lim3 and
all other progenitor HD proteins in that its expression is restricted to cells
in the pMN domain (Tanabe et al.,
1998
). Moreover, gain-of-function studies have provided evidence
that the ectopic expression of MNR2 in dorsal progenitor cells specifies many
aspects of motor neuron identity, while concomitantly suppressing spinal
interneuron fates (Tanabe et al.,
1998
).
After motor neurons have left the cell cycle, they acquire columnar subtype
identities that have classically been revealed by the position of motor neuron
cell bodies in the spinal cord and by the pattern of motor axon projections in
the periphery (Landmesser,
1978a; Landmesser,
1978b
; Tosney et al.,
1995
). Five major columnar groups of motor neuron can be
recognized on the basis of these criteria. Two of these groups are found
within the median motor column (MMC): a set of medial MMC neurons that is
generated at all rostrocaudal levels of the spinal cord and that extends axons
to axial muscles. At thoracic levels, a set of lateral MMC neurons is
generated that project their axons to body wall muscles
(Tosney et al., 1995
). A third
set, pre-ganglionic autonomic motor neurons [termed Column of Terni (CT)
neurons in chick], is also generated selectively at thoracic levels and these
neurons project axons to sympathetic neuronal targets
(Prasad and Hollyday, 1991
).
The final two columnar groups are found within the lateral motor column (LMC)
at limb levels of the spinal cord: medial LMC neurons project axons to
ventrally derived limb muscles and lateral LMC neurons project their axons to
dorsally derived limb muscles (Landmesser,
1978b
; Tosney et al.,
1995
).
Molecular insights into the specification of motor neuron columnar identity
have derived, in part, from the observation that each columnar subclass of
motor neurons is distinguishable by a distinctive profile of LIM HD
transcription factor expression (Tsuchida
et al., 1994; Ensini et al.,
1998
; Liu et al.,
2001
). Moreover, genetic studies in mice have begun to provide
evidence that the combinatorial expression of LIM HD proteins regulates the
subtype identity and connectivity of spinal motor neurons
(Shirasaki and Pfaff, 2002
).
For example, Isl1 function is required for the generation of all spinal motor
neurons (Pfaff et al., 1996
),
Lim3 (Lhx3) and Gsh4 (Lhx4) impose aspects of medial MMC identity
(Sharma et al., 1998
;
Sharma et al., 2000
), and the
expression of Lim1 by lateral LMC neurons establishes dorsal motor axonal
trajectories in the limb (Kania et al.,
2000
). The initial specification of lateral LMC neuronal identity
appears to be achieved by local retinoid signals provided by motor neurons
themselves, through the induction of Lim1 and the repression of Isl1
expression (Sockanathan and Jessell,
1998
). However, many of the extrinsic and intrinsic signaling
pathways that specify motor neuron columnar identity remain to be defined.
Some of the HD proteins expressed by motor neuron progenitors, notably Lim3
and Nkx6.1, continue to be expressed by subsets of postmitotic motor neurons
(Tsuchida et al., 1994;
Sharma et al., 1998
;
Cai et al., 2000
). One
potential strategy for assigning motor neuron columnar identity may,
therefore, involve the persistent expression of progenitor cell transcription
factors in subsets of postmitotic motor neurons. We show that expression of
the progenitor HD protein MNR2 persists in a subset of postmitotic motor
neurons, primarily those destined to populate the medial MMC, whereas its
expression is rapidly extinguished from CT neurons and most LMC neurons. By
contrast, HB9 is more widely expressed in postmitotic somatic motor neurons.
The differential expression of MNR2 and HB9 in postmitotic neurons appears to
contribute to the assignment of spinal motor neuron subtype identity. In
particular, the extinction of expression of MNR2 and HB9 from postmitotic
motor neurons is required for the generation of CT neurons. Moreover, the
action of Lim3 in suppressing CT generation
(Sharma et al., 2000
) appears
to be mediated through its ability to activate expression of MNR2 and HB9.
Thus, in neural progenitor cells, MNR2 appears to function in the initial
specification of motor neuron identity, whereas its later expression appears
to regulate motor neuron columnar subtype identity. In addition, our results
indicate that the ability of MNR2 to regulate motor neuron identity reflects
its role as a transcriptional repressor, providing further evidence for the
key role of transcriptional repression in motor neuron specification.
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MATERIALS AND METHODS |
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Recombinant retroviral vectors and expression constructs
MNR2 cDNA was isolated as described previously
(Tanabe et al., 1998), and a
MNR2
C-terminal construct was prepared by PCR-based cloning,
fusing amino acids 1-218 of MNR2 in frame with a series of five Myc-epitope
tags (Turner and Weintraub,
1994
). The MNR2 N-terminal deletion series was generated using a
PCR-based approach, adding a methionine to truncated MNR2 proteins. N-terminal
deletions of 5, 14, 27, 45 and 70 amino acid residues were generated, and
truncated sequences were cloned into RCASBP(B) constructs using a
SLAX shuttle vector (Hughes et al.,
1987
; Morgan and Fekete,
1996
). MNR2 HD constructs containing amino acids 146-218 were
fused to a series of Myc tags (MNR2 HD), to the Engrailed repressor domain
[MNR2-EnR (Smith and Jaynes,
1996
)], to the VP16 activation domain [MNR2-VP16
(Triezenberg et al., 1988
)] or
to amino acids 185-243 of E1a [MNR2-E1a
(Boyd et al., 1993
)]. A mutant
C-terminal domain of E1a (MNR2-E1amut) was prepared by PCR
mutagenesis of nucleotides encoding amino acids 235-237 of E1a to alanines
(PLDLS®PLAAA). A CMV enhancer, ß-actin promoter-based CAGGS plasmid
was used to express MNR2-EnR.
Immunocytochemistry and in situ hybridization histochemistry
RALDH2 was detected using a rabbit polyclonal antibody
(Sockanathan and Jessell,
1998). Monoclonal (4D5), rabbit polyclonal (K5) and guinea pig
polyclonal sera were used to detect Isl1/2 proteins
(Tsuchida et al., 1994
;
Tanabe et al., 1998
). Isl2 was
detected with monoclonal antibody (mAb) 4H9 and Isl1 was detected with rabbit
polyclonal sera A8 (Tsuchida et al.,
1994
); Lim1/Lim2 was detected with mAb 4F2 and rabbit antibody T2
(Tsuchida et al., 1994
); Lim3
was detected with mAb 4E12 (Ericson et
al., 1997
); and Chx10 was detected with a rabbit polyclonal serum
(Ericson et al., 1997
). Guinea
pig polyclonal serum was used to detect Olig2
(Novitch et al., 2001
). Rabbit
polyclonal serum was used to detect Irx3
(Novitch et al., 2001
) and a
monoclonal antibody was used to detect Nkx2.2
(Ericson et al., 1997
).
Chick embryos were fixed and prepared for immunocytochemistry as described
(Novitch et al., 2001).
Double- and triple-label analyses were perfomed with a BioRad 1024 confocal
microscope using Cy3-, Cy5- and FITC-conjugated secondary antibodies
(Jackson). ß-galactosidase staining was performed as described
(Kania et al., 2000
), in situ
hybridization was performed as described
(Schaeren-Wiemers and Gerfin-Moser,
1993
; Tsuchida et al.,
1994
) using MNR2, HB9, ChAT, BMP5, BMP4, and
ephrinA5 probes. BMP5 and BMP4 cDNAs were obtained
from Dr A. Kottman.
Gal4 transcription assay
Protein sequence N-terminal to the MNR2 HD was cloned into the Gal4
DNA-binding domain vector pSG424 (Sadowski
and Ptashne, 1989). COS-1 cells were co-transfected with pSG424
constructs, Gal4x5-E1b Luciferase and pRL-TK (Promega) plasmids using Fugene-6
lipofection reagent (Roche). Cells were harvested 48 hours later, and
luciferase activity measured using a Dual-Luciferase Assay Kit (Promega).
Gal4-luciferase activity was normalized to TK-Renilla luciferase activity.
Additional Gal4-constructs included pSG424-MyoD
(Weintraub et al., 1991
) and
pSG424-Engrailed repressor domain. Luciferase activity was compared with
values obtained with transfection of the Gal4 reporter plasmid alone.
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RESULTS |
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At stage 29, MNR2 expression was detected in medial MMC neurons at all
axial levels of the spinal cord (Fig.
1A,C,E). At thoracic levels of the spinal cord, MNR2 was expressed
in only a few lateral MMC neurons (Fig.
1C) and was absent from preganglionic autonomic motor neurons of
the Column of Terni (CT) (Fig.
1C; supplementary Fig. S1 at
dev.biologists.org/supplemental).
MNR2 was not expressed by hindlimb level lateral motor column (LMC) neurons
(Fig. 1E), and at forelimb
levels was expressed by only a few laterally positioned neurons within the LMC
(Fig. 1A). Many of these
laterally placed MNR2+ neurons co-expressed Lim3 (data not shown)
and appeared to correspond to rhomboideus motor neurons: a set of laterally
displaced medial MMC neurons (Tsuchida et
al., 1994) (data not shown).
|
Restrictions in the columnar identity of ectopic motor neurons
induced by MNR2
The persistence of MNR2 expression in medial MMC neurons raised the issue
of whether the ectopic motor neurons that are induced in more dorsal locations
in the spinal cord by MNR2 possess specific columnar identities. To assess
this, we induced ectopic dorsal motor neurons by electroporation of
MNR2 into the dorsal neural tube of stage 10-12 chick embryos,
permitting embryos to develop until stages 20-29 for analysis of generic and
columnar markers of motor neuron identity. Electroporation of MNR2 into one
side of the spinal cord (Fig.
2A) induced unilateral ectopic expression of four HD transcription
factors normally associated with motor neuron differentiation (Lim3, Isl1,
Isl2 and HB9) in neurons distributed along the dorsoventral axis of the spinal
cord (Fig. 2B-E) (see also
Tanabe et al., 1998). MNR2
also induced ectopic expression of the motor neuron Ig-family protein SC1
(Fig. 2F) and of the gene
encoding the acetylcholine synthetic enzyme choline acetyltransferase (ChAT)
(Tanabe et al., 1998
).
Moreover, the ectopic dorsal motor neurons induced by MNR2 projected axons out
of the spinal cord (supplementary Fig.
2 at
dev.biologists.org/supplemental).
Together, these findings show that MNR2 is an effective inducer of ectopic
motor neuron differentiation.
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Extinction of MNR2 expression is necessary for the specification of
CT identity
The rapid extinction of MNR2 and HB9 from CT neurons during normal
development raised the issue of whether downregulation of Mnx-class HD
proteins is required for the acquisition of CT columnar identity. To test
this, we electroporated MNR2 into the ventral neural tube at
prospective thoracic levels of stage 12-14 embryos and analyzed the pattern of
motor neuron generation at stages 27-29. Expression of MNR2 resulted in the
appearance of Isl1/2+ ectopic motor neurons in dorsal regions of
the spinal cord (Fig. 4A,B). In
addition, there was a striking loss of Isl1+ CT motor neurons that
are normally located in a dorsomedial position
(Fig. 4A,B). To test whether
the loss of these medially positioned Isl1+ motor neurons reflects
a failure in differentiation of CT neurons or simply a mispositioning, we
assayed the expression of BMP5, BMP4 and ephrinA5 after
MNR2 electroporation. Expression of BMP5, BMP4 and
ephrinA5 was lost on the electroporated side of the spinal cord
(Fig. 4C,D; data not shown).
Thus, maintaining expression of MNR2 in postmitotic motor neurons at thoracic
levels of the spinal cord prevents motor neurons from assuming a CT identity
(as assessed by their migratory route and profile of gene expression).
Expression of HB9 also resulted in the loss of dorsomedially positioned
Isl1+ motor neurons, and similarly inhibited expression of
BMP5, BMP4 and ephrinA5 (Fig.
4E-H; data not shown). These findings suggest that the extinction
of expression of Mnx class HD proteins is a prerequisite for postmitotic motor
neurons to progress to a CT identity.
|
We next addressed the issue of whether MNR2, HB9 and Lim3 act in parallel or sequential pathways to suppress the generation of CT neurons. We first examined whether misexpression of MNR2 within the normal domain of motor neuron generation maintains expression of Lim3 in postmitotic motor neurons. MNR2 was misexpressed at stages 12 to 14 and embryos were analyzed at stage 23, a time when BMP5 expression has normally been initiated, and MNR2 and HB9 expression extinguished from prospective CT neurons. Thoracic level motor neurons in which MNR2 expression was maintained lacked BMP5 expression (Fig. 5A,B), but did not exhibit prolonged expression of Lim3 or HB9 in prospective CT neurons (Fig. 5C-E). Similarly, prolonged expression of HB9 in thoracic level motor neurons failed to maintain expression of Lim3 or MNR2 (data not shown). Thus, the suppression of CT neuron specification by MNR2 and HB9 does not involve the maintenance of Lim3 expression in postmitotic motor neurons.
|
Together, these findings suggest that Lim3 suppresses CT identity indirectly, through the maintained expression of Mnx-class HD proteins, and also indicate that the downregulation of MNR2 and HB9 expression is a prerequisite for thoracic level motor neurons to progress to a CT identity.
Differential requirements for extinction of MNR2 and Lim3 expression
in the specification of LMC identity
At limb levels of the spinal cord, the expression of both MNR2 and Lim3 is
rapidly extinguished from LMC neurons, raising the question of whether the
downregulation of expression of both transcription factors is also required
for the specification of LMC identity. To test this, we misexpressed MNR2 or
Lim3 in motor neurons at forelimb and hindlimb levels of the spinal cord.
Maintained expression of MNR2 did not result in the upregulation of Lim3
expression in LMC neurons (Fig.
6A-C), nor did it eliminate two definitive markers of LMC neuronal
identity: RALDH2, and the co-expression of Isl2 and Lim1
(Fig. 6E-H). Similarly, we
found that maintained expression of HB9 did not induce Lim3
(Fig. 6D) or extinguish LMC
markers (data not shown). These findings are not unexpected given the
expression of HB9 by many LMC neurons during normal development and the
similar activities of MNR2 and HB9.
|
Evidence that MNR2 functions as a transcriptional repressor
Our findings, taken together with those of Tanabe et al.
(Tanabe et al., 1998),
indicate that MNR2 has three main activities in ventral neuronal
specification: (1) promoting the generation of motor neurons; (2) suppressing
ventral interneuron generation; and (3) suppressing the generation of CT
subtype identity. The ability of MNR2 to promote certain neuronal fates and
inhibit others raises the issue of whether its regulatory activities depend on
transcriptional activation or repression. To address this issue, we attempted
to identify functional domains of MNR2, monitoring activity on the basis of
its ability to induce ectopic Lim3 expression and to repress the
differentiation of CT neurons.
Expression of a form of MNR2 that lacks the entire C-terminal domain resulted in ectopic expression of Lim3 along the dorsoventral axis of the spinal cord, with a level of inductive activity similar to that of the wild-type protein (Fig. 7A,B). Thus, sequences C-terminal to the HD appear to be dispensable for the motor neuron inductive activity of MNR2. By contrast, deletion of the N-terminal 70 amino acids of MNR2 rendered the protein inactive (Fig. 7A). However, deletion of the N-terminal 44 or 28 amino acids of MNR2 resulted in only a partial reduction in the ability to activate expression of Lim3 (Fig. 7A). Deletion of the 14 N-terminal residues of MNR2 did not significantly reduce Lim3 inductive activity (Fig. 7A). These results suggest that motifs present in the domain between residues 15 and 70 of the N terminus of MNR2, in the context of the MNR2 HD, are necessary for motor neuron inductive activity.
To examine whether the N-terminal domain of MNR2 functions as a
transcriptional repressor or activator, we fused the entire N-terminal region
of MNR2 to the DNA-binding domain of the yeast transcription factor Gal4
(Gal4-MNR2N) and expressed this fusion protein in COS-1 cells,
together with a Gal4-UAS reporter construct
(Perlmann and Jansson, 1995).
Fusions of MyoD or the Drosophila Engrailed repressor (EnR) domain to
Gal4 provided controls for transcriptional activation and repression,
respectively (Fig. 7C). We
found that expression of the Gal4-MNR2N fusion protein repressed
transcription as efficiently as the Gal4-EnR protein
(Fig. 7C). Thus, the N-terminal
domain of MNR2 possesses transcriptional repressor activity in vitro, which
suggests that MNR2 functions as a repressor in its neural patterning
activities in vivo.
As most HD proteins that function as transcriptional repressors require the
recruitment of co-repressor proteins, we analyzed the N-terminal 70 residues
of MNR2 for potential co-repressor recruitment motifs. Analysis of the
N-terminal sequence of MNR2 and related Mnx family members revealed the
presence of a highly conserved 15 residue N-terminal domain
(Fig. 7D) that exhibits
sequence conservation with a motif initially characterized in the Engrailed
protein, termed the eh1 domain, which functions to recruit Groucho
co-repressor proteins (Smith and Jaynes,
1996
). However, as described above, a truncated MNR2 protein
lacking the N-terminal 14 amino acid eh1 motif is able to induce ectopic Lim3
expression with wild-type efficiency (Fig.
7A,E). Moreover, a fusion protein comprising the MNR2 HD fused to
the Engrailed eh1 domain had little or no Lim3 inductive activity
(Fig. 7A,F). These findings
raise doubts as to whether the motor neuron inductive activity of MNR2
requires the N-terminal eh1 domain or acts exclusively through recruitment of
Groucho class co-repressors.
In a search for additional structural motifs within the N-terminal domain
of MNR2 that might be required for its activity, we noted the presence of
motifs that resemble, albeit loosely, sequences required for recruitment of a
distinct class of transcriptional co-repressor, the Ctbp proteins
(Fig. 8A)
(Deltour et al., 2002;
Turner and Crossley, 2001
;
Chinnadurai, 2002
). The
N-terminal domains of Mnx class HD proteins in other organisms also possess
similar motifs (Fig. 8A). We
therefore considered whether fusion of the MNR2 HD to an efficient Ctbp
recruitment domain might mimic the activity of wild-type MNR2. We fused the
MNR2 HD to the C terminus of the oncoprotein E1a, which contains a potent Ctbp
co-repressor recruitment domain (Fig.
8B) (Boyd et al.,
1993
; Molloy et al.,
2001
). Ectopic expression of the MNR2 HD-E1a fusion protein
resulted in a potent induction of Lim3+ cells in dorsal regions of
the neural tube (Fig. 8C,D). In
addition, expression of the MNR2 HD-E1a fusion protein at thoracic levels
effectively suppressed the formation of CT neurons
(Fig. 8G,H). To test the
potential involvement of Ctbp recruitment in the activity of this fusion
protein we made use of the finding that the three C-terminal amino acids of
E1a are critical for Ctbp recruitment
(Turner and Crossley, 2001
;
Chinnadurai, 2002
). We
expressed a form of the MNR2 HD-E1a fusion that had been mutated at these
three residues (Fig. 8B) and
found that this protein did not induce Lim3 expression, nor did it repress the
generation of CT neurons (Fig.
8E,F; data not shown). Together, these findings support the idea
that MNR2 normally functions in motor neuron specification through its role as
a transcriptional repressor and raise the possibility that its repressor
activity may involve recruitment of Ctbp-like co-repressors.
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DISCUSSION |
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Accordingly, the differential extinction of transcription factor expression has a key role in the assignment of motor neuron columnar subtype identity (Fig. 9A). At the time of cell-cycle exit, all spinal motor neurons in chick express several HD transcription factors: Isl1 and Isl2, HB9, Lim3 and MNR2. The persistent expression of all five HD proteins is associated with the acquisition of medial MMC identity. The loss of Lim3 and MNR2 is associated with the emergence of lateral MMC and LMC identity, and the loss of MNR2, Lim3 and HB9 is required for the establishment of CT identity. Below, we discuss the role that the differential extinction of HD transcription factor expression plays in the progressive specification of motor neuron columnar subtype identity.
|
Our results provide evidence that the extinction of MNR2, HB9 and Lim3
expression is an essential step in the specification of CT identity
(Fig. 9A). Maintaining the
expression of any of these HD proteins in thoracic level motor neurons
prevents the molecular differentiation of CT neurons and eliminates their
dorsomedial migration. Furthermore, analysis of the hierarchical relationship
of these three HD proteins in postmitotic thoracic motor neurons indicates
that the early downregulation of Lim3 expression from prospective CT neurons
is required for the extinction of MNR2 and HB9. Thus, the suppression of CT
neuron specification observed after maintained expression of Lim3 is likely to
result from the persistence of expression of the Mnx class HD proteins MNR2
and HB9. Our findings on the specification of CT neuron identity also point to
a crucial difference in the hierarchical relationship between MNR2 and Lim3 in
motor neuron progenitor cells and postmitotic motor neurons. In progenitor
cells, MNR2 activates Lim3 but not vice versa
(Tanabe et al., 1998), whereas
in postmitotic motor neurons Lim3 activates MNR2 but not vice versa. These
findings suggest that the nature of interactions between HD transcription
factors changes dramatically with the transition from motor neuron progenitors
to postmitotic motor neurons.
However, the hierarchical relationship between MNR2, Lim3 and HB9 that
emerges from the analysis of motor neuron columnar specification at thoracic
levels of the spinal cord does not extend to limb levels. Although MNR2 is
also extinguished from LMC neurons, expression of the related Mnx class
protein HB9 is maintained in LMC neurons
(Fig. 9A). Consistent with this
observation, maintained expression of MNR2 does not inhibit the
differentiation of LMC neurons. By contrast, maintained expression of Lim3
effectively represses the generation of LMC neurons (see also
Sharma et al., 2000). Thus,
despite their similar patterns of expression in motor neuron subsets, MNR2 and
Lim3 appear to have distinct roles in the assignment of motor neuron columnar
identity.
The restriction of MNR2 expression to medial MMC neurons observed during
normal development appears to fit with the columnar subtype identity of motor
neurons induced by ectopic expression of MNR2. Many of the MNR2-induced
ectopic motor neurons express Lim3, which is consistent with the view that
MNR2 expression is associated with motor neurons of a medial MMC-like
identity. Moreover, at thoracic levels of the spinal cord, postmitotic motor
neurons induced by MNR2 express HB9 and fail to express BMP5,
consistent with the role of MNR2 in repressing CT neuronal differentiation.
Moreover, at limb levels of the spinal cord, motor neurons induced by MNR2
fail to express RALDH2 and do not co-express Isl2 and Lim1. Thus, MNR2-induced
motor neurons also fail to exhibit LMC character. These findings with MNR2
overexpression at limb levels contrast with results of notochord grafting at
limb levels of the spinal cord, where ectopic dorsal LMC neurons are generated
(Fukushima et al., 1996).
Together, these findings point to a Shh-induced program of LMC neuronal
differentiation that is not recruited by MNR2 expression alone.
Evolutionary conservation and divergence in Mnx class homeodomain
protein function
One additional issue that emerges from our findings on the actions of MNR2
in motor neuron specification in chick concerns the apparent absence of a
corresponding MNR2 gene in mammals
(Arber et al., 1999;
Thaler et al., 1999
). Of
relevance here, is the finding that the bHLH protein Olig2 has a pattern of
expression and role in motor neuron specification similar to that invoked for
MNR2 (Novitch et al., 2001
;
Mizuguchi et al., 2001
;
Zhou et al., 2000
;
Zhou and Anderson, 2002
;
Lu et al., 2002
). Olig2 is an
efficient inducer of MNR2, Lim3 and Isl1 expression in chick spinal cord and
is required for motor neuron generation in vivo
(Novitch et al., 2001
;
Mizuguchi et al., 2001
;
Zhou and Anderson, 2002
;
Lu et al., 2002
;
Takebayashi et al., 2002
). As
the combined expression of Lim3 and Isl1 is sufficient to induce HB9
expression (Tanabe et al.,
1998
; Thaler et al.,
2002
), it seems as if multiple transcriptional pathways converge
at the point of regulation of Lim3 and Isl1 expression during the
specification of motor neuron fate. The apparent absence of an MNR2
counterpart in mouse implies that the Olig2-dependent, MNR2-independent
pathway is the primary route of motor neuron generation in mammals, whereas in
chick both Olig2 and MNR2 have the ability to trigger Lim3 and Isl1 expression
and motor neuron generation. Nevertheless, both Olig2 and MNR2 appear to
function as transcriptional repressors in the pathway of motor neuron
specification (Novitch et al.,
2001
) (Fig. 9B).
Thus, the activation of Lim3 and Isl1 expression by MNR2 and Olig2 is likely
to result from the repression of a common repressor of genes involved in motor
neuron generation (Fig.
9B).
HB9, a close relative of MNR2, is expressed in motor neurons in all
vertebrate species examined (Pfaff et al.,
1996; Saha and Grainger, 1997;
Tanabe et al., 1998
;
Arber et al., 1999
;
Thaler et al., 1999
), raising
the additional issue of whether HB9 might assume the functions of MNR2 in
mouse. In chick, HB9 is confined to postmitotic motor neurons, and thus its
actions are likely to be restricted to the control of later aspects of motor
neuron differentiation. Moreover, in mouse, HB9 is also confined largely to
postmitotic motor neurons (Arber et al.,
1999
; Thaler et al.,
1999
), and inactivation of HB9 does not impair the
initial generation of motor neurons. Thus, the mouse HB9 protein is unlikely
to have acquired the progenitor cell functions performed by MNR2 in chick
(Tanabe et al., 1998
).
However, the loss of HB9 expression in mouse results in the persistent
expression of Lhx3 (Lim3) and the related LIM HD protein Lhx4 in many or all
postmitotic motor neurons (Arber et al.,
1999
; Thaler et al.,
1999
). Thus, one possible function of HB9 in LMC and lateral MMC
neurons may be to ensure the rapid extinction of Lim3 expression after
cell-cycle exit. The deregulation of Lim3 expression observed in HB9
mutants, could, however, also reflect the misassignment of motor neuron and V2
interneuron identity, as these interneurons also express Lim3
(Arber et al., 1999
;
Thaler et al., 1999
;
Thaler et al., 2002
).
Nevertheless, these findings, taken together with the results of MNR2
overexpression in chick, support the idea that Mnx class HD proteins have
significant roles in assigning motor neuron columnar subtype identity in
vertebrates. These conclusions have also received support from recent
observations that HB9 has a critical role in motor neuron specification in
Drosophila (Broihier and Skeath,
2002
).
Motor neuron specification by transcriptional repression
Several lines of evidence suggest that MNR2, and its relative HB9, function
as transcriptional repressors during the process of motor neuron
specification. First, the N-terminal domain of MNR2 essential for its activity
in motor neuron specification can function as a potent transcriptional
repressor in cell-based reporter assays. Second, the HD of MNR2, when fused to
a known co-repressor recruitment domain, the E1a C-terminal domain
(Chinnadurai, 2002; Turner and
Crossley, 2002), can mimic the activity of the wild-type MNR2 protein, both in
motor neuron specification and in repression of CT subtype identity. These
findings are complemented by genetic studies of HB9 function in mouse, in
which HB9 has been shown to repress its own expression
(Arber et al., 1999
;
Thaler et al., 1999
) and to
repress expression of V2 interneuron determinants in motor neurons
(Tanabe et al., 1998
;
Thaler et al., 2002
).
The precise mechanism of MNR2- and HB9-mediated transcriptional repression
remains unclear. MNR2, like many other HD proteins
(Muhr et al., 2001), possesses
a well conserved eh1 motif that, in other contexts, can recruit Groucho class
co-repressors (Smith and Jaynes,
1996
). However, elimination of the eh1 motif in MNR2 does not
abolish its ability to induce motor neuron generation. Moreover, fusion of the
HD of MNR2 to a potent Groucho recruitment domain results in poor motor
neuron-inducing activity in vivo. Thus, the repressor functions of MNR2, and
by inference of HB9, may not simply reflect the recruitment of Groucho class
co-repressors. Our data show that the MNR2 HD-E1a C-terminal repressor domain
fusion protein mimics the activity of the wild-type MNR2 protein, raising the
possibility that MNR2 repressor activity involves the recruitment of Ctbp
class co-repressors (Turner and Crossley,
2001
; Chinnadurai,
2002
). However, additional experiments are necessary to resolve
whether the repressor functions of MNR2 normally involve the recruitment of
Ctbp class co-repressors. In addition, studies on co-repressor function in
Drosophila raise the possibility of cooperative interactions between
eh1 Groucho recruitment and Ctbp recruitment domains present within the same
transcription factor (Hasson et al.,
2001
; Barolo et al.,
2002
).
Regardless of the precise co-repressors recruited by MNR2, our evidence
supports the view that MNR2 function in vivo is likely to reflect its role as
a transcriptional repressor. These findings therefore add to the emerging view
that the logic of motor neuron fate specification is grounded in
transcriptional repression (Muhr et al.,
2001). Many of the progenitor transcription factors involved in
motor neuron specification at steps upstream of MNR2, e.g. Nkx6.1, Nkx6.2 and
Olig2, also function as transcriptional repressors
(Muhr et al., 2001
;
Novitch et al., 2001
;
Vallstedt et al., 2001
).
Unlike the Nkx6 and Mnx proteins, Olig2 does not possess a clear eh1 motif,
further supporting the idea that the transcriptional repressors that function
in motor neuron specification recruit distinct classes of co-repressor
protein. Finally, the similarities in sequence and activities of Mnx class HD
proteins, and genetic studies of HB9 in mouse and Drosophila indicate
that all Mnx class proteins may function as transcriptional repressors
(Arber et al., 1999
;
Thaler et al., 1999
;
Broihier and Skeath, 2002
). As
HB9 expression in spinal cord is restricted largely to postmitotic motor
neurons, these observations imply that the key role of transcriptional
repression in motor neuron fate specification extends from progenitor cells
into postmitotic neurons.
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ACKNOWLEDGMENTS |
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Footnotes |
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