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First published online 4 December 2008
doi: 10.1242/dev.028043
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The Solomon Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 N Wolfe Street, Baltimore, MD 21205, USA.
Author for correspondence (e-mail:
ssockan1{at}jhmi.edu)
Accepted 7 November 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Retinoids, Nolz1, Grg5, Motoneuron, Identity, Repressor
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Vallstedt et al., 2001).
However, neurons retain a degree of plasticity in their fates after cell cycle
exit, implying that the induction or maintenance of cell fate-specification
programs in postmitotic neurons is crucial for the acquisition and
preservation of final neuronal identity
(Gross et al., 2002;
Moran-Rivard et al., 2001;
Muller et al., 2002;
Pearson and Doe, 2004). While
the mechanisms that regulate progenitor patterning and differentiation are
emerging, less is known about the molecular pathways that shape and maintain
identity in postmitotic neurons.
The study of spinal motoneuron (MN) development has helped elucidate the
transcriptional mechanisms that generate and consolidate postmitotic neuronal
fate (Jessell, 2000). Spinal
MNs derive from a discrete ventral progenitor domain termed pMN. In the chick,
MNR2 is expressed in the S phase of the terminal cell cycle of pMN progenitors
and triggers the expression of a cascade of factors essential for somatic MN
development (Tanabe et al.,
1998). All newly differentiating MNs coexpress Islet1 and Lim3,
two transcription factors crucial for maintaining somatic MN properties.
Islet1 is required for MN survival and, together with Lim3, regulates the
expression of HB9, a homeodomain (HD) protein necessary for suppressing
intrinsic interneuron specification programs in developing MNs
(Pfaff et al., 1996;
Arber et al., 1999;
Thaler et al., 1999;
Thaler et al., 2002). These
`generic' MNs subsequently diversify into distinct neuronal subtypes that are
manifest by the organization of their cell bodies into specific motor columns,
their characteristic axonal projection patterns, and their distinct LIM-HD
protein expression profiles (Landmesser,
1978; Tosney et al.,
1995; Tsuchida et al.,
1994; Jessell,
2000). MNs forming the medial division of the median motor column
(MMCm) span all rostral-caudal levels and innervate axial muscles, whereas the
preganglionic MNs of the column of Terni (CT) and MNs of the lateral MMC
(MMCl) are found at thoracic regions
(Prasad and Hollyday, 1991;
Jessell, 2000). At limb levels,
lateral motor column (LMC) MNs that innervate the limb form medial and lateral
divisions, which innervate ventrally and dorsally derived limb muscles,
respectively. MN diversification is first apparent by the downregulation of
Lim3 in prospective CT, MMCl and LMC MNs
(Sharma et al., 2000). Within
Lim3- MNs, cross-repressive interactions between different Hox HD
proteins consolidate rostralcaudal Hox protein distributions, which activate
the expression of distinct LIM-HD proteins that dictate MN subtype identity
and connectivity (Liu et al.,
2001; Dasen et al.,
2003; Sharma et al.,
2000; Kania et al.,
2000). For example, cross-repressive interactions between Hoxc9
and Hoxc6 define their expression domains at thoracic and forelimb levels,
where they respectively regulate the formation of CT and MMCl, and of forelimb
LMC columnar identities (Dasen et al.,
2003).
Retinoic acid (RA) signaling pathways play central roles in regulating
generic MN differentiation and in the specification and maintenance of
forelimb LMC identity and lateral LMC divisional character
(Solomin et al., 1998;
Sockanathan and Jessell, 1998;
Diez del Corrall et al., 2003;
Novitch et al., 2003;
Sockanathan et al., 2003;
Vermot et al., 2005;
Ji et al., 2006). RA signals
directly regulate gene transcription through the activity of nuclear
receptors; however, the factors that mediate RA responses to regulate
postmitotic MN identity remain unknown
(Maden, 2002). One candidate
is Nlz2/Nolz1, a member of the Noc, Elbow and Tlp-1 (NET) family of atypical
zinc-finger-containing transcriptional regulators
(Nakamura et al., 2004;
Runko and Sagerstrom, 2004;
Hoyle et al., 2004). Both
zebrafish Nlz2 (also known as Znf503 - ZFIN) and its mouse homolog Nolz1 (also
known as Zfp503 - Mouse Genome Informatics) are expressed at known sites of RA
synthesis and function in the brain (Runko
and Sagerstrom, 2004; Hoyle et
al., 2004; Chang et al.,
2004). Nlz2 is found in the hindbrain, where it is required for
the specification of rhombomeric identity, a process known to be dependent
upon RA signaling, whereas Nolz1 is coincidently expressed with Raldh3 in the
lateral ganglionic eminence (LGE), a region of the telencephalon that gives
rise to the striatum. Nolz1 expression in cell culture is RA-inducible, a
finding supported by the identification of a functional direct repeat 5
retinoic acid response element (DR5-RARE) upstream of the Nolz1
translational start site (Chang et al.,
2004). Based on these collective observations, we examined if and
how Nlz2/Nolz1 mediates RA-dependent events regulating postmitotic MN
development.
Here, we show that Nolz1 regulates the diversification of Lim3-
motor columns from Lim3+ MNs, consolidates their formation and
controls the specification and diversification of LMC MNs by regulating Hox
and LIM-HD protein expression. These divergent functions of Nolz1 require
distinct repressor activities that depend in part on modulatory functions of
the Gro-TLE protein, Grg5 (Fisher and
Caudy, 1998). This study thus reveals that RA signals regulate the
progressive specification of postmitotic MN columnar and divisional identity
by inducing a single pivotal molecule that executes multiple
fate-specification transcriptional programs through the assembly of
functionally distinct repressor complexes.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) for
in ovo electroporation (William et al.,
2003). For siRNA experiments, Nolz1 and Grg5 siRNA duplexes were
electroporated as previously described
(Rao et al., 2004). Target
sequences are as follows: Nolz1, 5'-AAACTTGCTCGCAGATAGGAA-3' and
5'-AATTGCTATGATTGTATCGTA-3'; Grg5,
5'-GCGAGAAATCGGAGATGCA-3' and
5'-TGGCCAAGGAGGACAAGAA-3'. DsRed siRNA sequences are as published
(Rao et al., 2004). Luciferase
assays were carried out as described
(Novitch et al., 2001;
Nishihara et al., 2003).
Immunohistochemistry and in situ hybridization
Chicken embryos were prepared for immunohistochemistry and in situ
hybridization as described (Sockanathan
and Jessell, 1998). Primary antibodies used were as follows.
Polyclonal rabbit antiserum against chick Nolz1 was generated by immunizing
rabbits with a GST fusion of the first 373 amino acids of Nolz1 (Covance).
Primary antibodies were used at the following dilutions: rabbit anti-Nolz1,
1:1000; K5 (rabbit anti-Isl1/2), 1:2500; T4 (rabbit anti-Lim1/2), 1:3000;
guinea pig anti-Isl1/2, 1:10,000 (provided by T. M. Jessell, HHMI, Colombia
University, New York, NY); monoclonal antibodies 4F2 (anti-Lim1/2), 1:2; 4H9
(anti-Isl2), 1:100; 674E12 (anti-Lim3), 1:100; 81.5C10 (anti-HB9/MNR2), 1:100
(Developmental Studies Hybridoma Bank); rabbit anti-MNR2, 1:8000 (provided by
B. Novitch, UCLA, Los Angeles, CA); goat anti-Hoxc6 (provided by J. Dasen,
Smilow Institute, New York, NY); rabbit anti-Lhx3 (Abcam), 1:500; goat
anti-β-galactosidase (Arnel), 1:3000; GFP, mouse anti-SC1, 1:40; rabbit
anti-Chx10, 1:2000; mouse anti-En1 (4G11), 1:100; mouse anti-Evx1/2
(99.1-32A), 1:50; and mouse anti-HA (12CA5), 1:1000.
Images were captured using a Zeiss LSM 5 Pascal confocal microscope. In situ hybridization was performed as described (Schaeren-Wiemers and Gerfin-Moser, 1993). Quantitation of neuronal number was carried out using ten sections per embryo from four to ten embryos.
Yeast two-hybrid screen
Yeast two-hybrid screens were carried out using the Matchmaker Two-Hybrid
System (Clontech). The library was prepared from RNA generated from St 23
chick spinal cords and cotransformed into yeast strain AH109 with the GAL4
activation domain plasmid pGADT7-Rec and bait construct pGBKT7-Nolz1. Only
colonies growing on SD/-Ade/-His/-Leu/-Trp/X-
-Gal selective medium were
picked for further analysis.
Co-immunoprecipitation
Flag or HA epitope tags were fused to the N-terminus of Nolz1 or Grg5 and
cloned into pCAGGS or pCS2 vectors. Transiently transfected HEK293T cells were
harvested and homogenized in lysis buffer using standard procedures. Lysates
were precleaned by incubation with GammaBind G Sepharose beads (GE Healthcare)
followed by centrifugation, and were mixed with anti-Flag M2 (Sigma)-bound
beads overnight at 4°C under constant rotation. After extensive washing,
the precipitated proteins were analyzed by SDS-PAGE and western blot using
anti-HA antibodies (Santa Cruz).
Reverse northern blot analysis
Two different chick Nolz1 EST clones (University of Delaware Chick EST
Database) were electrophoresed in agarose gels and blotted according to
standard procedures. Filters were probed with cDNAs derived from Hamburger
Hamilton St 19 brachial chick neural explants grown for 18 hours in the
absence or presence of retinol. Retinol was used to take advantage of the
endogenous expression of the retinoid synthetic enzyme Raldh2, which is
expressed in brachial MNs. Retinol is metabolized within the explants to
substrates for Raldh2, which subsequently catalyzes the formation of
endogenous retinoid metabolites
(Sockanathan and Jessell,
1998). RNA from 100 explants was isolated using Trizol (Gibco
BRL). cDNAs were generated and amplified using the Marathon cDNA PCR
Amplification Kit (Stratagene).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). To
test whether RA signals can regulate Nolz1 expression in the chick spinal
cord, we performed reverse northerns. Densitometry analysis showed a 5-fold
increase of Nolz1 expression in explants exposed to retinol
(Fig. 1B). Consistent with a
role for RA signals in regulating Nolz1 expression, electroporation
of constructs expressing RAR403, a potent dominant-negative retinoid receptor
that disrupts RA signaling, caused a decrease in Nolz1 expression in the
dorsal spinal cord (Fig. 1C,D)
(Novitch et al., 2003;
Sockanathan et al., 2003).
Nolz1 is expressed in subsets of developing spinal MNs
To examine the spatial and temporal distribution of Nolz1 in the chick
spinal cord, we carried out a developmental time course of Nolz1 mRNA
expression. Prior to St 24, MNs at all rostral-caudal levels express
Nolz1; however, from St 25 onwards, Nolz1 is restricted to a
subset of laterally located forelimb and lumbar MNs
(Fig. 2A-H; data not shown). To
determine whether Nolz1 expression correlates with the development of specific
motor columns, we examined the expression of Nolz1 protein in relation to
molecular markers that define the major spinal motor columns and divisions
(Tsuchida et al., 1994). All
newly differentiated spinal MNs coexpress Islet1 and Lim3. Lim3 expression is
maintained in MMCm neurons spanning all axial levels, but it is rapidly
downregulated in LMC MNs at limb levels, and in CT and MMCl neurons at
thoracic regions of the spinal cord
(Tsuchida et al., 1994). Nolz1
expression was initiated in a narrow band of laterally located
Islet1+ Lim3+ neurons at St 18, but rapidly segregated
with forelimb and thoracic Islet1/2+ MNs that had downregulated
Lim3 expression (Fig.
2I,J,N,O). By St 24, no MNs coexpressed Nolz1 and Lim3, and there
was a clear demarcation between Nolz1- Lim3+ and
Nolz1+ Lim3- MNs at all axial levels
(Fig. 2K,P). MMCm MNs marked by
Islet1/2 and Lim3 coexpression do not express Nolz1
(Fig. 2K,P; data not shown). By
contrast, Nolz1 expression at limb levels was confined to medial
(Islet1/2+, Lim3-, Lim1-) and lateral
(Lim3-, Islet2+, Lim1+) divisions of the LMC,
while at thoracic regions, Nolz1 was detected in MMCl neurons
(Islet1/2+, HB9+, Lim3-) and prospective CT
MNs (Islet1/2+, HB9-, Lim3-)
(Fig. 2K,L,P,Q; data not
shown). From St 25 onwards, Nolz1 expression was downregulated in thoracic
MNs; however, at limb levels, Nolz1 expression was maintained in lateral LMC
(LMCl) neurons and in a subset of medial LMC (LMCm) neurons
(Fig. 2M,R; data not shown).
Thus, Nolz1 expression initiates in a narrow band of newly differentiated
Islet1+ Lim3+ MNs, rapidly segregates with
Lim3- motor columns at all axial levels, and subsequently localizes
to subsets of limb-level LMC neurons (Fig.
2S).
|
). Plasmids
expressing GFP were coelectroporated to mark the transfected side
(Fig. 3A,F). Electroporated
embryos showed a 
50% reduction of Nolz1 mRNA and protein when compared
with the contralateral non-electroporated side, or when electroporated with
unrelated siRNAs (Fig.
3A,B,F,G; data not shown; see Fig. S1 in the supplementary
material). However, no changes in the number of Lim3+ MMCm MNs at
either forelimb or thoracic levels of the spinal cord were observed,
consistent with the lack of Nolz1 expression in MMCm MNs
(Fig. 3C,E,H,J).
Lim3- MNs give rise to medial and lateral divisions of the LMC at
forelimb levels, while at thoracic regions they generate MMCl and CT motor
columns (Tsuchida et al.,
1994; Jessell,
2000). At forelimb levels, knockdown of Nolz1 resulted in
a 30% loss of LMCm neurons and a 70% loss of LMCl cells, as compared with the
contralateral non-electroporated side or when control unrelated siRNAs were
electroporated, while at thoracic levels there was a reduction of CT and MMCl
MNs by 50% and 40%, respectively (Fig.
3D,E,I,J). The percentage decrease in CT and MMCl MNs correlates
well with the extent of Nolz1 knockdown; at forelimb levels, however,
LMCm generation appeared less affected by the knockdown of Nolz1 than
neurons of the LMCl subtype. This difference might reflect the relative
distribution of Nolz1 in LMC neurons, in that all LMCl neurons, but only a
subset of LMCm neurons, normally express Nolz1
(Fig. 2M). These results
suggest that Nolz1 is specifically required for the formation of
Lim3- motor columns, consistent with its early expression in
Lim3- MN populations in the spinal cord.
Grg5 interacts with Nolz1
Nolz1 contains a single, atypical zinc finger and is thus unlikely to bind
DNA directly; however, NET proteins can activate or repress transcription
through the formation of multimeric complexes
(Nakamura et al., 2004;
Runko and Sagerstrom, 2004).
We reasoned that it would be more informative to evaluate the role of Nolz1 in
Lim3- motor column development by investigating its function in the
absence and presence of its associated proteins. To identify proteins that
interact with Nolz1, we performed a yeast two-hybrid screen using full-length
Nolz1 fused to the GAL4 DNA-binding domain as bait, together with a cDNA
library generated from St 23 ventral chick spinal cords fused to the GAL4
activation domain. We identified one clone that overlapped with Nolz1
expression in chick spinal MNs (Fig.
4A). This clone corresponded to Grg5, a member of the Gro-TLE
family of co-repressors that functions as a `derepressor' of other Groucho
(Grg) proteins owing to its inability to complex with histone deacetylases
(HDACs) (Fisher and Caudy,
1998; Brantjes et al.,
2001). The overlap of Grg5 and Nolz1 expression
in the ventral spinal cord suggests that Grg5 is a viable candidate for
mediating Nolz1 function in spinal MNs. We confirmed that Grg5 interacts with
Nolz1 by co-immunoprecipitation (co-IP) assays using extracts prepared from
HEK293T cells transfected with tagged versions of Nolz1 and Grg5
(Fig. 4B,C). To identify the
Grg5 interaction sites on Nolz1, we generated a series of deletions within
Nolz1 and examined which of these deletions lacked the ability to interact
with Grg5 by co-IP (Fig. 4C).
Deletion of a putative Grg consensus binding site (FKPY, 
161-170)
within Nolz1 did not abolish the interaction between Nolz1 and Grg5
(Fig. 4B,C). Instead, deletion
of the C-terminal 22 amino acids significantly reduced Nolz1-Grg5 complex
formation (Nolz1C22) (Fig.
4B,C). Together, these results show that Grg5 can specifically
interact with the C-terminus of Nolz1.
Nolz1 repressor function is modulated by Grg5 activity
To investigate the transcriptional properties of Nolz1, we assayed its
function using an in vitro reporter-based assay. Full-length Nolz1 was fused
to the GAL4 DNA-binding domain (GalNolz1) and cotransfected into COS-7 cells
with a reporter plasmid containing GAL4 DNA-binding sequences cloned upstream
of a basal E1B promoter and the luciferase reporter gene
(GAL4x5-E1b-luciferase) (Novitch et al.,
2001). Transfection of GAL4x5-E1b-luciferase into COS-7 cells
generated basal transcriptional activity that was repressed 11-fold by
GalNolz1 (Fig. 4D).
Cotransfection of Grg5 expression plasmids caused a reproducible decrease in
Nolz1-dependent repressor activity (Fig.
4D). Consistent with this function, transfection of
Nolz1C22 fused to the GAL4 DNA-binding domain resulted in a marked
increase of Nolz1 repressor activity (GalNolz1C22;
Fig. 4D). To determine the
transcriptional properties of Nolz1 and Grg5 in vivo, we repeated these
experiments using extracts prepared from dissected chick spinal cords
electroporated with the same series of expression plasmids
(Nishihara et al., 2003).
However, in this case we utilized MH100, which contains GAL4 DNA-binding
sequences upstream of a minimal thymidine kinase promoter and the luciferase
reporter gene (Muhr et al.,
2001). Similar to the assays carried out in COS-7 cells, GalNolz1
repressed basal MH100 expression (Fig.
4E). But, coelectroporation of Grg5 caused a marked `derepression'
of GalNolz1-dependent transcriptional activity
(Fig. 4E). GalNolz1C22
showed increased repressor activity compared with GalNolz1, consistent with
the ability of Grg5 to modulate Nolz1 repressor function.
|
;
Brantjes et al., 2001;
Muhr et al., 2001).
Nolz1 overexpression leads to the downregulation of Lim3 and to MN loss
To investigate how Nolz1 regulates the formation of Lim3- MNs,
we overexpressed Nolz1 under the control of a 3 kb promoter derived from the
mouse Hb9 (Mnx1 - Mouse Genome Informatics) gene, which
initiates expression in newly differentiating MNs (HB9-Nolz1)
(Lee et al., 2004). Embryos
electroporated with plasmids expressing GFP under the Hb9 promoter
(HB9-GFP) expressed Lim3 in 80% of electroporated MNs
(Fig. 5D,G). By contrast, less
than 5% of electroporated MNs expressing Nolz1 were Lim3+,
suggesting that Nolz1 is sufficient to cause the downregulation of Lim3
expression (Fig. 5A-C,G).
Although the Nolz1-expressing cells had initiated MN differentiation, which
was evident by the induction of Nolz1 expression from the Hb9
promoter, they lacked Islet1/2 and HB9 expression
(Fig. 5B,C; data not shown).
Furthermore, a number of the cells were labeled by TUNEL, indicating that they
had initiated apoptosis (data not shown).
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C22 expression into chick spinal cords.
Quantification of the percentage of Lim3+ cells expressing Nolz1
and Nolz1C22 showed that removal of the Grg5-interaction domain
resulted in a minor increase in the number of Nolz1 and Lim3 coexpressing
cells, but did not restore Lim3 coexpression to control levels
(Fig. 5E,G). This observation
suggests that the Nolz1-dependent repression of Lim3 expression does not
require its interaction with Grg5. In vivo transcriptional assays indicated
that Nolz1 functions as a transcriptional repressor
(Fig. 5E). To confirm that
Nolz1 downregulates Lim3 through repressor activity and not through activator
functions derived from interactions with unidentified binding partners in
vivo, we fused the VP16 activator domain to the C-terminus of full-length
Nolz1 (Nolz1-VP16). We verified that Nolz1-VP16 functions as an activator
using in vitro and in vivo transcriptional reporter assays
(Fig. 4D,E). HB9-Nolz1-VP16
constructs failed to downregulate Lim3 expression in electroporated chick
spinal cords (Fig. 5F,G). These
collective observations indicate that Nolz1 repressor activity downregulates
Lim3 expression in spinal MNs and that this repressor function is not
dependent on its association with Grg5. These findings suggest that Nolz1
repressor function plays crucial roles in the early segregation of
Lim3- and Lim3+ MN populations by its ability to
suppress Lim3 expression in postmitotic MNs.
Nolz1 and Grg5 coexpression induces ectopic MNs
The loss of Lim3- motor columns in Nolz1 knockdown
embryos is unlikely to result from Lim3 derepression, as forced expression of
Lim3 in MNs is not detrimental to MN survival
(Sharma et al., 2000;
William et al., 2003). These
observations suggest additional roles for Nolz1 in regulating Lim3-
motor column development that are distinct from its function in downregulating
Lim3 expression. MN generation and maintenance are orchestrated by key HD
transcription factors that include MNR2, Lim3, Islet1 and HB9
(Pfaff et al., 1996;
Tanabe et al., 1998;
Sharma et al., 1998;
Arber et al., 1999;
Thaler et al., 1999;
William et al., 2003;
Thaler et al., 2002). We
reasoned that Nolz1 might function to maintain the expression of these key
transcription factors in prospective Lim3- MNs. If so, then Nolz1
should be capable of regulating their transcription, and thus might induce
their expression in gain-of-function assays. We tested this possibility by
expressing Nolz1 in the dorsal spinal cord by in ovo electroporation of Nolz1
expression constructs driven by the chick β-actin promoter
(pCAGGS-Nolz1).
Electroporation of Nolz1 alone did not lead to the ectopic expression of MN
factors dorsally (see Fig. S2 in the supplementary material). By contrast,
coexpression of Nolz1 and Grg5 induced the cell-autonomous expression of Lim3,
Islet1 and HB9 (Fig. 6A-D). A
small number of cells expressed Islet2 2 days after electroporation,
reflecting the temporal profile of Islet2 expression in mature MNs (data not
shown) (Tsuchida et al.,
1994). Nolz1 and Grg5 coexpression failed to induce MNR2,
consistent with the restriction of endogenous Nolz1 and Grg5 expression to
postmitotic MNs (Fig. 4A,
Fig. 6C). Consistent with
previous reports, Grg5 overexpression alone did not lead to ectopic MNR2, HB9,
Islet1, Islet2 or Lim3 expression (see Fig. S2 in the supplementary material)
(Muhr et al., 2001).
Coelectroporation of Nolz1C22 and Grg5 did not elicit the ectopic
expression of MN HD proteins, suggesting that Nolz1-Grg5 complex formation is
necessary for these events (Fig.
6E,F; data not shown). Furthermore, Nolz1-VP16 expression did not
induce Lim3, HB9, Islet1 or Islet2, consistent with the ability of Nolz1-Grg5
complexes to implement MN determinant expression through repressor activities
(Fig. 6G,H).
HB9 expression is directly induced by Islet1-Lim3 multimers, and HB9 can
upregulate the expression of Islet1 and Lim3
(Arber et al., 1999;
Thaler et al., 1999;
Thaler et al., 2002;
William et al., 2003). To
examine the hierarchy of MN determinant expression induced by Nolz1-Grg5, we
compared the expression profiles of these markers relative to each other.
Nolz1 and Grg5 coexpression induced many HB9+ cells in the absence
of Lim3 or Islet1, suggesting that Nolz1-Grg5 provides an alternative pathway
to upregulate HB9 expression that does not require Islet1 and Lim3
(Fig. 6B-D). Furthermore, 35%
of ectopic Islet1+ cells and 62% of ectopic Lim3+ cells
lacked detectable HB9 expression (Fig.
6B,D; n=126 Islet1+ cells, n=42
Lim3+ cells analyzed). Taken together, these results suggest that
Nolz1-Grg5 complexes can independently upregulate the expression of HB9,
Islet1 and Lim3.
|
|
). In
support of their MN identity, none of the ectopic Islet1+ cells
expressed the V0 or V1 interneuron markers Evx1/2 or En1
(Fig. 6L,M)
(Pierani et al., 1999).
However, 1-2 ectopic cells expressed the V2 marker Chx10, probably owing to
the absence of HB9, which is required to repress V2-specific fates in MNs
(Fig. 6K)
(Arber et al., 1999;
Thaler et al., 1999). To
determine whether Nolz1-Grg5-induced MNs adopt a Lim3- columnar
identity, we examined electroporated embryos 48 hours after coelectroporation
of Nolz1 and Grg5. We found that 98% of ectopic Islet1+ MNs lacked
Lim3 expression (n=120 Islet1+ cells analyzed). Thus, Nolz1/Grg5 repressor activity can initiate MN differentiation programs downstream of MNR2, resulting in MNs with a Lim3- expression profile. The transient expression of Lim3 mediated by Nolz1 and Grg5 coexpression reflects the profile of Nolz1 expression in developing spinal MNs, where Nolz1 is coexpressed with Lim3 in a lateral band of newly generated MNs, prior to its rapid segregation with Lim3- spinal MNs (Fig. 2S).
Grg5 is required for Lim3- motor column development
Our gain-of-function studies suggest that Grg5 plays central roles in
regulating the development and formation of Lim3- motor columns. To
examine whether Grg5 is necessary for Lim3- motor column
development, we electroporated 21 bp siRNAs designed against the Grg5
ORF into chick embryonic spinal cords at St 14, and analyzed electroporated
embryos at St 23/24 (Rao et al.,
2004). Grg5 mRNA levels were efficiently decreased in
siRNA-treated embryos; however, owing to the lack of available antibodies
against chick Grg5, we were unable to assess the efficiency of reducing Grg5
protein (Fig. 7A,B).
Nevertheless, we evaluated embryos electroporated with Grg5 siRNAs for the
formation of Lim3+ MMCm MNs and Lim3- LMCl and LMCm MNs
at forelimb levels of the spinal cord. No changes in the number of
Lim3+ MMCm MNs were detected in embryos that showed efficient
knockdown of Grg5 mRNA. By contrast, we consistently detected a 30%
reduction in the number of Lim3- LMCl and LMCm MNs, as compared
with the non-electroporated contralateral side, or when control siRNAs were
electroporated (Fig. 7C-E).
These results indicate that Grg5 is required for the formation of
Lim3- MNs, and further supports our model that Nolz1-Grg5 complexes
regulate the generation of Lim3- motor columns in the spinal
cord.
|
). We first examined the onset of Nolz1 expression in
prospective forelimb LMC neurons in relation to that of Hoxc6, a key
postmitotic determinant of forelimb LMC identity
(Dasen et al., 2003). At St 17,
we detected many Nolz1+ MNs at forelimb levels, but very few,
weakly expressing Hoxc6+ cells
(Fig. 8A,B). By St 19, many
Nolz1+ MNs were distributed along the mediolateral axis, a lateral
subset of which coexpressed Hoxc6 (Fig.
8C). These data indicate that Nolz1 expression initiates prior to
the induction of Hoxc6 in prospective forelimb LMC neurons.
To test whether Nolz1 or Grg5 is capable of inducing Hoxc6 expression, we
electroporated pCAGGS-Nolz1 into chick spinal cords and analyzed dorsal
thoracic spinal cords at St 24, when Hoxc6 is normally absent. Expression of
Nolz1 or Grg5 alone failed to elicit Hoxc6 expression (see Fig. S2 in the
supplementary material). By contrast, coelectroporation of Nolz1 and Grg5
induced many dorsal Hoxc6+ cells, and upregulated Hoxc6 expression
in a small number of thoracic MNs (Fig.
8D,E,H,I). Notably, dorsal Hoxc6 expression rarely coincided with
cells coexpressing Grg5 and Nolz1, implying that Nolz1/Grg5-dependent
induction of Hoxc6 occurs non-cell-autonomously. Grg5 and Nolz1C22
coexpression did not induce Hoxc6, suggesting that Nolz1 and Grg5 interact to
upregulate Hoxc6 expression (Fig.
8F; see Fig. S2 in the supplementary material). Electroporation of
Nolz1-VP16 failed to elicit Hoxc6 expression
(Fig. 8G). Taken together,
these findings suggest that Nolz1-Grg5 complexes induce Hoxc6 expression
through transcriptional repressor functions, and provide a mechanism that
links Hoxc6-dependent specification of forelimb LMC identity with RA signaling
pathways.
|
|
; Kania et al.,
2000; Vermot et al.,
2005; Ji et al.,
2006). Nolz1 and Grg5 coexpression induces Lim3- MNs in
the dorsal spinal cord (Fig.
6). To examine whether a subset of these neurons acquires lateral
LMC fates, we examined embryos 2 days after coelectroporation with Nolz1 and
Grg5. LMCl neurons are distinguished by their coexpression of Islet2 and Lim1,
and by their downregulation of Islet1
(Tsuchida et al., 1994).
Sixty-three percent of ectopic Islet1/2+ MNs coexpressed Lim1 when
embryos were coelectroporated with Nolz1 and Grg5
(Fig. 8J,K; n=374
Islet1/2+ cells). However, staining with an Islet2-specific
antibody showed that the majority of these cells did not express Islet2,
demonstrating that they had induced Lim1 expression but failed to downregulate
Islet1 (Fig. 8K,L). Ectopic
Islet1/2+ cells did not express interneuron markers such as Evx1/2,
En1 and Chx10, but were ChAT+ and SC1+
(Fig. 6I-M). These observations
suggest that Nolz1 and Grg5 coexpression results in Islet1+
Islet2- MNs that had initiated a partial LMCl specification
program. Hoxc6 triggers a temporal sequence of LMC formation, whereby
Raldh2-derived RA induces LMCl subtype identity
(Sockanathan and Jessell,
1998; Dasen et al.,
2003; Vermot et al.,
2005). Nolz1 and Grg5 coexpression does not induce dorsal Raldh2
expression, suggesting that Lim1 induction in ectopic MNs arises independently
of Raldh2 function (data not shown). Moreover, many dorsal Lim1+
MNs did not coexpress Hoxc6 and overexpression of Hoxc6 in the dorsal spinal
cord did not induce Islet1/2+ Lim1+ cells
(Fig. 8M; see Fig. S3 in the
supplementary material). Taken together, these results argue that Nolz1 can
induce MNs that are capable of initiating partial programs of LMCl
specification downstream of Hoxc6 and Raldh2 activity. |
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Nolz1 regulates the segregation and development of Lim3- MNs
We show here that Nolz1 is sufficient to downregulate Lim3 expression in
postmitotic MNs by Grg5-independent repressor functions. Lim3 downregulation
is an essential prerequisite for the acquisition of LMC, CT and MMCl columnar
fates, as sustained Lim3 expression suppresses CT and LMC motor column
formation, and causes MNs to adopt cell body settling positions, gene
expression profiles and axonal projections characteristic of MMCm MNs
(Sharma et al., 2000;
William et al., 2003).
Previous studies have shown that HB9 also functions to suppress Lim3
expression (Arber et al., 1999;
Thaler et al., 1999). We find
that Nolz1 can upregulate HB9 expression through a separate repressor activity
involving association with Grg5. Thus, our studies support a model in which
Nolz1 implements at least two pathways that converge to control the timely
repression of Lim3 expression in prospective LMC, CT and MMCl MNs. These
findings invoke crucial roles for Nolz1 in regulating the early segregation of
Lim3- motor columns from Lim3+ MMCm MNs.
Ablating Nolz1 causes the loss of Lim3- MNs, suggesting
that Nolz1 has additional functions in the development and survival of CT,
MMCl and LMC motor columns besides downregulating Lim3 expression. We find
that Nolz1-Grg5 repressor complexes can induce terminally differentiated MNs
with a Lim3- molecular identity, through upregulating a series of
key HD transcription factors. Since endogenous Nolz1 expression initiates in
postmitotic MNs and not in progenitor cells, this finding is consistent with
the model that Nolz1-Grg5 complexes implement HD-dependent transcriptional
programs that are necessary for consolidating and/or maintaining the
properties and survival of postmitotic Lim3- MNs. Accordingly,
Nolz1-Grg5 complexes are not sufficient to activate MNR2 expression,
consistent with their role in MN consolidation or maintenance rather than
induction (Tanabe et al.,
1998). The involvement of Nolz1-Grg5 repressor complexes in this
regard parallels the requirement for Nkx-Grg complexes in derepressing MN
differentiation programs during MN generation
(Muhr et al., 2001). These
observations suggest that continuous derepression strategies are required in
progenitors and postmitotic MNs to generate and actively preserve the
properties and survival of postmitotic MNs. The function of Nolz1-Grg5
complexes might differ between limb-level and thoracic motor columns. The
prolonged expression of Nolz1 at limb levels suggests that Nolz1-Grg5
complexes play more sustained roles in the specification and maintenance of
LMC neurons as compared with CT and MMCl neurons, in which Nolz1 is
downregulated after St 24. Indeed, prolonged retinoid receptor activation is
detrimental to thoracic MN survival, suggesting that retinoid-dependent
pathways operate transiently during thoracic motor column development
(Sockanathan et al.,
2003).
Nolz1 regulates forelimb LMC specification
Disrupting RA signals in prospective forelimb MNs causes putative LMC MNs
to adopt thoracic identities, suggesting that RA signals have dual roles in
specifying LMC fates and suppressing thoracic-specific columnar
differentiation programs (Sockanathan et
al., 2003). We show that Nolz1-Grg5 repressors induce Hoxc6, which
can drive LMC-specific programs of differentiation and suppress thoracic
columnar identity in limb-level MNs (Liu
et al., 2001; Dasen et al.,
2003). These findings provide a mechanism that links RA signaling
with the specification of forelimb LMC identity through the Nolz1-dependent
induction of Hoxc6 expression. How do Nolz1-Grg5 repressor complexes
upregulate Hoxc6? Hoxc9 can repress Hoxc6 expression cell-autonomously in
spinal MNs (Dasen et al.,
2003). However, our results argue against the possibility that
Nolz1 derepresses Hoxc6 expression through inhibition of Hoxc9, as Nolz1
functions non-cell-autonomously to upregulate Hoxc6. Instead, Nolz1 might
induce Hoxc6 through the derepression of additional signaling pathways that
remain to be identified.
A second question is how Nolz1-Grg5 complexes might regulate the restricted
expression of Hoxc6 at forelimb levels, when Nolz1 and Grg5 are expressed at
all axial levels at the time of Hoxc6 induction. One option is that the
intermediary components that relay Nolz1-Grg5 effects are restricted primarily
to spinal MNs at forelimb levels. In support of this idea, very few thoracic
MNs induce Hoxc6 when Nolz1 is overexpressed, as compared with those in the
dorsal spinal cord. Alternatively, the restricted expression of Hoxc6 by
Nolz1-Grg5 activity might depend on the function of other Hox proteins. Hoxc9
is induced by FGFs and is expressed in thoracic progenitors and spinal MNs
(Dasen et al., 2003). Thus,
early Hoxc9 expression in progenitors and MNs could override the later
inductive capabilities of Nolz1-Grg5 complexes, thereby repressing Hoxc6 at
thoracic levels. This Hox-dependent mechanism could potentially operate at
lumbar regions via Hox10 proteins, thus confining the ability of Nolz1-Grg5
complexes to induce Hoxc6 expression to forelimb levels of the spinal cord
(Dasen et al., 2003;
Wu et al., 2008).
Nolz1 induces Lim1 expression in prospective lateral LMC neurons
Our expression analyses and functional experiments collectively support a
role for Nolz1 in lateral LMC neuronal development. Ectopic MNs induced by
Nolz1-Grg5 complexes coexpress Lim1, a LIM-HD protein that partly defines LMCl
molecular identity and directs their dorsal axonal trajectory
(Tsuchida et al., 1994;
Kania et al., 2000). However,
the Lim1+ MNs induced by Nolz1-Grg5 complexes coexpress Islet1,
which is normally downregulated in LMCl neurons
(Tsuchida et al., 1994). These
observations indicate that Nolz1-Grg5 complexes implement a subset of LMCl
differentiation pathways, specifically those regulating LMCl axonal
projections. They also imply that Lim1 induction can be uncoupled from the
downregulation of Islet1, suggesting that independent pathways function to
execute these previously linked events. We show that the Lim1+ MNs
induced by Nolz1-Grg5 complexes are generated independently of prior Raldh2
and Hoxc6 function. These results support a mechanism whereby Nolz1-Grg5
repressors function downstream of forelimb LMC specification and Raldh2
activity, to induce Lim1 expression in prospective LMCl MNs. Nolz1 is
expressed in LMCl neurons until at least St 29, suggesting that Nolz1-Grg5
complexes might function to maintain Lim1 expression in LMCl cells.
In conclusion, our study suggests that RA signals specify multiple events governing MN subtype identity through the induction of Nolz1. Nolz1 exhibits strong transcriptional repressor activity, and this repressor function is modulated by interactions with Grg5. Importantly, Nolz1 repressor complexes are functionally distinct, suggesting that their individual components differ. Notably, our observation that Grg5 modulates Nolz1 repressor activity invokes the possibility that graded Nolz1 repressor activities are a crucial component in implementing selective transcriptional pathways that direct different aspects of MN development. The induction of a single key factor with functional flexibility, such as Nolz1, circumvents the need to generate large numbers of proteins that individually execute separate developmental pathways, and provides a useful and effective strategy to regulate diverse events in neuronal development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/2/231/DC1
|
Footnotes |
|---|
* Present address: Department of Pharmacology, Weill Medical College of
Cornell University, 1300 York Avenue, Box 70, New York, NY 10065, USA 
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Arber, S., Han, B., Mendelsohn, M., Smith, M., Jessell, T. M.
and Sockanathan, S. (1999). Requirement for the homeobox gene
Hb9 in the consolidation of MN identity. Neuron
23,659
-674.[CrossRef][Medline]
Brantjes, H., Roose, J., van de Wetering, M. and Clevers, C.
(2001). All Tcf HMG box transcription factors interact with
Groucho-related co-repressors. Nucleic Acids Res.
29,1410
-1419.
Chang, C. W., Tsai, C. W., Wang, H. F., Tsai, H. C., Chen, H.
Y., Tsai, T. F., Takahashi, H., Li, H. Y., Fann, M. J., Yang, C. W. et al.
(2004). Identification of a developmentally regulated
striatum-enriched zinc-finger gene, Nolz-1, in the mammalian brain.
Proc. Natl. Acad. Sci. USA
101,2613
-2618.
Dasen, J. S., Liu, J. P. and Jessell, T. M.
(2003). MN columnar fate imposed by sequential phases of Hox-c
activity. Nature 425,926
-933.[CrossRef][Medline]
Diez del Corral, R., Olivera-Martinez, I., Goriely, A., Gale,
E., Maden, M. and Storey, K. (2003). Opposing FGF and
retinoid pathways control ventral neural pattern, neuronal differentiation,
and segmentation during body axis extension. Neuron
40, 65-79.[CrossRef][Medline]
Fisher, A. L. and Caudy, M. (1998). Groucho
proteins: transcriptional corepressors for specific subsets of DNA-binding
transcription factors in vertebrates and invertebrates. Genes
Dev. 12,1931
-1940.
Gross, M. K., Dottori, M. and Goulding, M.
(2002). Lbx1 specifies somatosensory association interneurons in
the spinal cord. Neuron
34,535
-549.[CrossRef][Medline]
Hoyle, J., Tang, Y. P., Wielllette, E. L., Wardle, F. C. and
Sive, H. (2004). nlz gene family is required for hindbrain
patterning in the zebrafish. Dev. Dyn.
229,835
-846.[CrossRef][Medline]
Jessell, T. M. (2000). Neuronal specification
in the spinal cord: inductive signals and transcriptional codes.
Nat. Rev. Genet. 1,20
-29.[CrossRef][Medline]
Ji, S. J., Zhuang, B., Falco, C., Schneider, A.,
Schuster-Gossler, K., Gossler, A. and Sockanathan, S. (2006).
Mesodermal and neuronal retinoids regulate the induction and maintenance of
limb innervating spinal MNs. Dev. Biol.
297,249
-261.[CrossRef][Medline]
Kania, A., Johnson, R. L. and Jessell, T. M.
(2000). Coordinate roles for LIM homeobox genes in directing the
dorsoventral trajectory of motor axons in the vertebrate limb.
Cell 102,161
-173.[CrossRef][Medline]
Landmesser, L. (1978). The distribution of
motoneurones supplying chick hindlimb muscles. J.
Physiol. 284,371
-389.
Lee, S. K., Jurata, L. W., Funahashi, J., Ruiz, E. C. and Pfaff.
S. L. (2004). Analysis of embryonic motoneuron gene
regulation: derepression of general activators function in concert with
enhancer factors. Development
131,3295
-3306.
Liu, J. P., Laufer, E. and Jessell, T. M.
(2001). Assigning the positional identity of spinal MNs:
rostrocaudal patterning of Hox-c expression by FGFs, Gdf11, and retinoids.
Neuron 32,997
-1012.[CrossRef][Medline]
Maden, M. (2002). Retinoid signalling in the
development of the central nervous system. Nat. Rev.
Neurosci. 3,843
-853.[CrossRef][Medline]
Moran-Rivard, L., Kagawa, T., Saueressig, H., Gross, M. K.,
Burrill, J. and Goulding, M. (2001). Evx1 is a postmitotic
determinant of v0 interneuron identity in the spinal cord.
Neuron 29,385
-399.[CrossRef][Medline]
Muhr, J., Andersson, E., Persson, M., Jessell, T. M. and
Ericson, J. (2001). Groucho-mediated transcriptional
repression establishes progenitor cell pattern and neuronal fate in the
ventral neural tube. Cell
104,861
-873.[CrossRef][Medline]
Muller, T., Brohmann, H., Pierani, A., Heppenstall, P. A.,
Lewin, G. R., Jessell, T. M. and Birchmeier, C. (2002). The
homeodomain protein lbx1 distinguished two major programs of neuronal
differentiation in the dorsal spinal cord. Neuron
34,551
-562.[CrossRef][Medline]
Nakamura, M., Runko, A. P. and Sagerstrom, C. G.
(2004). A novel family of zinc finger genes involved in embryonic
development. J. Cell. Biochem.
93,887
-895.[CrossRef][Medline]
Nishihara, S., Tsuda, L. and Ogura, T. (2003).
The canonical Wnt pathway directly regulates NRSF/REST expression in chick
spinal cord. Biochem. Biophys. Res. Commun.
7, 55-63.
Novitch, B. G., Chen, A. I. and Jessell, T. M.
(2001). Coordinate regulation of MN subtype identity and
pan-neuronal properties by the bHLH repressor Olig2.
Neuron 31,773
-789.[CrossRef][Medline]
Novitch, B. G., Wichterle, H., Jessell, T. M. and Sockanathan,
S. (2003). A requirement for retinoic acid-mediated
transcriptional activation in ventral neural patterning and MN specification.
Neuron 40,81
-95.[CrossRef][Medline]
Pearson, B. J. and Doe, C. Q. (2004).
Specification of temporal identity in the developing nervous system.
Annu. Rev. Cell Dev. Biol.
20,619
-647.[CrossRef][Medline]
Pfaff, S. L., Mendelsohn, M., Stewart, C. L., Edlund, T. and
Jessell, T. M. (1996). Requirement for LIM homeobox gene Isl1
in MN generation reveals a MN-dependent step in interneuron differentiation.
Cell 84,309
-320.[CrossRef][Medline]
Pierani, A., Brenner-Morton, S., Chiang, C. and Jessell, T.
M. (1999). A sonic hedgehog-independent, retinoid-activated
pathway of neurogenesis in the ventral spinal cord.
Cell 97,903
-915.[CrossRef][Medline]
Prasad, A. and Hollyday, M. (1991). Development
and migration of avian sympathetic preganglionic neurons. J. Comp.
Neurol. 307,237
-258.[CrossRef][Medline]
Rao, M. and Sockanathan, S. (2005).
Transmembrane protein GDE2 induces MN differentiation in vivo.
Science 309,2212
-2215.
Rao, M., Baraban, J., Rajaii, F. and Sockanathan, S.
(2004). In vivo comparative study of RNAi methodologies by in ovo
electroporation in the chick embryo. Dev. Dyn.
231,592
-600.[CrossRef][Medline]
Runko, A. P. and Sagerstrom, P. (2004).
Isolation of nlz2 and characterization of essential domains in Nz family
proteins. J. Biol. Chem.
279,11917
-11925.
Shaeren-Wiemers, N. and Gerfin-Moser, A.
(1993). A single protocol to detect transcripts of various types
and expression levels in neural tissue and cultured cells: in situ
hybridization using digoxigenin-labelled cRNA probes.
Histochemistry 100,431
-440.[CrossRef][Medline]
Sharma, K., Sheng, H. Z., Lettieri, K., Li, H., Karavanov, A.,
Potter, S., Westphal, H. and Pfaff, S. L. (1998). LIM
homeodomain factors Lhx3 and Lhx4 assign subtype identities for MNs.
Cell 95,817
-828.[CrossRef][Medline]
Sharma, K., Leonard, A. E., Lettieri, K. and Pfaff, S. L.
(2000). Genetic and epigenetic mechanisms contribute to MN
pathfinding. Nature 406,515
-519.[CrossRef][Medline]
Sockanathan, S. and Jessell, T. M. (1998).
MN-derived retinoid signaling specifies the subtype identity of spinal MNs.
Cell 94,503
-514.[CrossRef][Medline]
Sockanathan, S., Perlmann, T. and Jessell, T. M.
(2003). Retinoid receptor signaling in postmitotic MNs regulates
rostrocaudal positional identity and axonal projection pattern.
Neuron 40,97
-111.[CrossRef][Medline]
Solomin, L., Johansson, C. B., Zetterstrom, R. H., Bissonette,
R. P., Heyman, R. A., Olson, L., Lendahl, U., Frisen, J. and Perlmann, T.
(1998). Retinoid X receptor signaling in the developing spinal
cord. Nature 395,398
-402.[CrossRef][Medline]
Tanabe, Y., William, C. and Jessell, T. M.
(1998). Specification of MN identity by the MNR2 homeodomain
protein. Cell 95,67
-80.[CrossRef][Medline]
Thaler, J. P., Harrison, K., Sharma, K., Lettieri, K., Kehrl, J.
and Pfaff, S. L. (1999). Active suppression of interneuron
programs within developing MNs revealed by analysis of homeodomain factor HB9.
Neuron 23,675
-687.[CrossRef][Medline]
Thaler, J. P., Lee, S. K., Jurata, L. W., Gill, G. N. and Pfaff,
S. L. (2002). LIM factor Lhx3 contributes to the
specification of MN and interneuron identity through cell-type-specific
protein-protein interactions. Cell
110,237
-249.[CrossRef][Medline]
Tosney, K. W., Hotary, K. B. and Lance-Jones, C.
(1995). Specifying the target identity of motoneurones.
BioEssays 17,379
-382.[CrossRef][Medline]
Tsuchida, T. N., Ensini, M., Morton, S. B., Baldessare, M.,
Edlund, T., Jessell, T. M. and Pfaff, S. L. (1994).
Topographic organization of embryonic MNs defined by expression of LIM
homeobox genes. Cell 79,957
-970.[CrossRef][Medline]
Vallstedt, A., Muhr, J., Pattyn, A., Pierani, A., Mendelsohn,
M., Sander, M., Jessell, T. M. and Ericson, J. (2001).
Different levels of repressor activity assign redundant and specific roles to
Nkx6 genes in MN and interneuron specification. Neuron
31,743
-755.[CrossRef][Medline]
Vermot, J., Schuhbaur, B., Le Mouellic, H., McCaffery, P.,
Garnier, J. M., Hentsch, D., Brulet, P., Niederreither, K., Chambon, P.,
Dolle, P. and Le Roux, I. (2005). Retinaldehyde dehydrogenase
2 and Hoxc8 are required in the murine brachial spinal cord for the
specification of Lim1+ motoneurons and the correct distribution of Islet1+
motoneurons. Development
132,1611
-1621.
William, C., Tanabe, Y. and Jessell, T. M.
(2003). Regulation of MN subtype identity by repressor activity
of Mnx class Homeodomain proteins. Development
130,1523
-1536.
Wu, Y., Wang, G., Scott, S. A. and Capecchi, M. R.
(2008). Hoxc10 and Hoxd10 regulate mouse columnar, divisional and
motor pool identity of lumbar motoneurons. Development
135,171
-182.
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