RESEARCH ARTICLE
231
Development 136, 231-240 (2009) doi:10.1242/dev.028043
Nolz1 is induced by retinoid signals and controls
motoneuron subtype identity through distinct repressor
activities
Sheng-Jian Ji*, Goran Periz and Shanthini Sockanathan†
The acquisition and maintenance of final neuronal identity depends in part upon the implementation of fate-specification
programs in postmitotic neurons; however, the mechanisms involved remain unclear. In the developing spinal cord, retinoic acid
(RA) signaling pathways specify the columnar and divisional identities of postmitotic motoneurons (MNs). Here we show that RA
signals induce expression of the NET transcriptional regulator Nolz1 in differentiated chick MNs, where it regulates the progressive
specification of prospective Lim3-negative motor columns. Nolz1 controls the initial formation of forelimb and thoracic Lim3negative motor columns by downregulating Lim3 expression and maintaining the expression of key homeodomain proteins
necessary for MN identity and survival. At forelimb levels, Nolz1 specifies lateral motor column (LMC) identity by inducing the
expression of the postmitotic LMC determinant Hoxc6, and implements the partial specification of lateral LMC identity through
Lim1 induction. The specificity of Nolz1 function depends upon distinct repressor activities that require, in part, the modulatory
activity of Grg5, an atypical member of the Gro-TLE family of co-repressors. Thus, RA signals regulate diverse events in MN subtype
specification by inducing the expression of a key transcriptional regulator that controls multiple developmental pathways via
functionally distinct repressor complexes.
INTRODUCTION
The establishment of a functional nervous system depends upon the
coordinated generation and maintenance of diverse neuronal
subtypes during embryonic and postnatal development. In
progenitor cells, cross-repressive transcriptional mechanisms elicit
the expression of distinct transcription factors that confer subsequent
neuronal fate (Jessell, 2000; 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; MoranRivard 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
The Solomon Snyder Department of Neuroscience, The Johns Hopkins University
School of Medicine, 725 N Wolfe Street, Baltimore, MD 21205, USA.
*Present address: Department of Pharmacology, Weill Medical College of Cornell
University, 1300 York Avenue, Box 70, New York, NY 10065, USA
†
Author for correspondence (e-mail: [email protected])
Accepted 7 November 2008
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
DEVELOPMENT
KEY WORDS: Retinoids, Nolz1, Grg5, Motoneuron, Identity, Repressor
RESEARCH ARTICLE
member of the Noc, Elbow and Tlp-1 (NET) family of atypical zincfinger-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 RAinducible, 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 GroTLE 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.
MATERIALS AND METHODS
In ovo electroporation and luciferase transcriptional assays
All cDNAs were cloned into pCAGGS or a 3 kb Hb9 promoter-based
vector (Lee et al., 2004) 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.
Development 136 (2)
Yeast two-hybrid screen
Yeast two-hybrid screens were carried out using the Matchmaker TwoHybrid 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/Xα-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).
RESULTS
Nolz1 expression is induced by RA in the
developing spinal cord
We cloned the chick homolog of Nolz1 by RT-PCR using RNA
prepared from dissected Hamburger Hamilton stage (St) 21-23 chick
spinal cords. The predicted Nolz1 protein shares similar domains
with zebrafish, mouse and human Nolz1 homologs, and is 74% and
78% identical to zebrafish and mouse Nolz1, respectively (Fig. 1A)
(Nakamura et al., 2004). 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
DEVELOPMENT
232
Fig. 1. Nolz1 expression is induced by RA signals in spinal cord
explants. (A) Conserved domains between chick (c), mouse (m),
zebrafish (z) and human (h) predicted Nolz1 ORFs. Sp, Sp1-related
domain; FKPY, putative Groucho consensus binding site; Btd,
Drosophila Buttonhead domain; ZF, zinc-finger domain. Numbers
indicate percentage identity in the regions between these domains.
(B) Reverse northern of two different chick Nolz1 EST clones (EST1,
EST2) probed with pools of cDNAs generated from St 19 ventral chick
embryonic spinal cord explants grown in the presence (+) or absence (–)
of retinol (Rol). c29.1 is a cDNA fragment from an unrelated gene
known to be transcriptionally unresponsive to RA signals (Rao and
Sockanathan, 2005). (C,D) Confocal images of chick spinal cords
electroporated with RAR403 showing decreased dorsal Nolz1
expression (asterisk). Motoneurons are reduced as their generation is
dependent on RA signaling.
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).
Nolz1 loss-of-function results in reduced numbers
of Lim3– spinal MNs
To define the requirement for Nolz1 in MN development, we ablated
Nolz1 expression using short interfering RNAs (siRNAs).
Oligonucleotides (21 bp) designed against the Nolz1 ORF and
3⬘UTR were electroporated into chick spinal cords at St 12, prior to
the onset of Nolz1 expression and MN generation (Rao et al., 2004).
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
RESEARCH ARTICLE
233
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 nonelectroporated 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, Δ161170) 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 (Nolz1ΔC22)
(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
DEVELOPMENT
Nolz1 regulates motoneuron identity
234
RESEARCH ARTICLE
Development 136 (2)
Fig. 2. Nolz1 is expressed in subsets of postmitotic
spinal MNs. (A-H) In situ hybridization of Nolz1 mRNA
expression on transverse sections of embryonic chick
spinal cords. (I-R) Confocal micrographs showing
analysis of Nolz1 protein expression as compared with
molecular markers of specific spinal motor columns.
Arrows mark Nolz1 and Lim3 coexpressing MNs.
(S) Summary of Nolz1 expression in relation to motor
column development. The dashed orange line indicates
transient Nolz1 expression. MMC, median motor
column; MMCm, medial division of the MMC; MMCl,
lateral division of the MMC; LMC, lateral motor
column; LMCm, medial division of the LMC; LMCl,
lateral division of the LMC; CT, column of Terni.
Taken together, these data suggest that Nolz1 functions as a
repressor molecule, and that Grg5 interactions with Nolz1 serve to
modulate Nolz1 repressor function. Mechanistically, Grg5 is known
to modulate Grg co-repressor activity, raising the possibility that
classical Grg proteins mediate Nolz1-dependent transcriptional
repression (Fisher and Caudy, 1998; 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 (HB9Nolz1) (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
DEVELOPMENT
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 Nolz1ΔC22 fused to the GAL4 DNA-binding domain resulted in a
marked increase of Nolz1 repressor activity (GalNolz1ΔC22; 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).
GalNolz1ΔC22 showed increased repressor activity compared with
GalNolz1, consistent with the ability of Grg5 to modulate Nolz1
repressor function.
Nolz1 regulates motoneuron identity
RESEARCH ARTICLE
235
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).
To examine whether the ability of Nolz1 to downregulate Lim3
expression utilizes endogenous Grg5 activity, we electroporated
Hb9 promoter constructs driving Nolz1ΔC22 expression into
chick spinal cords. Quantification of the percentage of Lim3+ cells
expressing Nolz1 and Nolz1ΔC22 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-offunction 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 Nolz1ΔC22 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 Nolz1Grg5 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
DEVELOPMENT
Fig. 3. Nolz1 ablation causes a decrease of Lim3– motor columns. (A-D,F-I) Confocal micrographs of sectioned St 23/24 chick spinal cords
electroporated with Nolz1 siRNAs and examined for Nolz1 expression and motor column markers by immunohistochemistry at forelimb (FL) and
thoracic (T) levels of the spinal cord. (E,J) Bar charts showing the percentage of MNs corresponding to different motor columns in electroporated
Nolz1 siRNAs (black bars), control siRNAs (gray bars) and non-electroporated contralateral (white bars, set as 100%) sides of the spinal cord at
forelimb (E) and thoracic (J) regions. n=4-5 embryos, mean ± s.e.m.
RESEARCH ARTICLE
Fig. 4. Nolz1 and Grg5 interact and function as transcriptional
repressors. (A) In situ hybridization of Grg5 mRNA expression in
transverse sections of chick embryonic spinal cords. (B) Coimmunoprecipitation (IP) analyses of Grg5 protein interactions with Nolz1.
(C) Schematic of the Nolz1 coding region. FKPY, putative Grg consensus
binding site; Btd, Buttonhead domain; ZF, atypical zinc-finger domain.
Lines beneath indicate the regions deleted within the Nolz1 coding
sequence; the numbers indicate the corresponding amino acids that are
deleted. Whether Nolz1 retains (+) or loses (–) its ability to interact with
Grg5 in co-IP is indicated alongside. (D) Luciferase-based transcriptional
assays using extracts from transfected COS-7 cells. Data are from three
replicate experiments, mean ± s.e.m. Student’s t-test compared with
Nolz1: Nolz1+Grg5, P=0.0055; Nolz1ΔC22, P=0.0016. (E) Luciferasebased transcriptional assays using extracts from electroporated chick
spinal cords. n=6-8 embryos, mean ± s.e.m. Student’s t-test compared
with Nolz1: Nolz1+Grg5, P<0.000001; Nolz1ΔC22, P=0.0029. Compared
with GAL4-only: Nolz1+Grg5, P=0.0003.
Lim3+ cells analyzed). Taken together, these results suggest that
Nolz1-Grg5 complexes can independently upregulate the expression
of HB9, Islet1 and Lim3.
Dorsal cells that express the MN HD transcription factors when
Nolz1 and Grg5 are coexpressed consistently occupied lateral
positions, suggesting that the induction of these HD proteins
occurred in the context of normal neurogenesis (Fig. 6A-D). Ectopic
cells expressed choline acetyltransferase (ChAT), the enzyme
responsible for acetylcholine synthesis, and the MN surface marker
SC1, consistent with their adopting MN fates (Fig. 6I,J) (Tanabe et
al., 1998). 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
Development 136 (2)
Fig. 5. Nolz1 represses Lim3 expression in postmitotic MNs.
(A-F) Confocal micrographs of St 21 chick spinal cord sections
electroporated on the right. The high levels of Nolz1 elicited by
overexpression preclude the detection of endogenous Nolz1 in the
spinal cord under these imaging conditions. (G) Bar chart quantifying
the percentage of electroporated cells expressing Lim3. n=10 embryos,
mean ± s.e.m.
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
DEVELOPMENT
236
Nolz1 regulates motoneuron identity
RESEARCH ARTICLE
237
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.
Nolz1 interacts with Grg5 to induce Hoxc6
expression in the spinal cord
The retention of Nolz1 expression in LMC neurons suggests that
Nolz1 might be required for the development of LMC motor columns.
Since RA signaling pathways are necessary for specifying forelimb
LMC identity, we examined whether Nolz1 mediates the
transcriptional programs required for triggering forelimb LMC
specification (Sockanathan et al., 2003). 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 Nolz1ΔC22 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
DEVELOPMENT
Fig. 6. Nolz1-Grg5 complexes induce postmitotic
MNs. Transverse sections of St 21 embryonic chick
spinal cords electroporated on the right. White
brackets denote ectopic MNs.
(A-H,J-M) Immunohistochemical analyses using
antibodies against MN and ventral interneuron
markers. Arrows indicate ectopic MNs expressing the
MN surface marker SC1 (J) or Chx10 (K). (I) Arrows
mark ectopic MNs expressing transcripts for choline
acetyltransferase (ChAT). Dashed lines mark the lateral
extent of the spinal cord.
238
RESEARCH ARTICLE
Development 136 (2)
Fig. 7. Grg5 knockdown decreases the number of Lim3– motor
columns. (A-D) St 24 chick embryos electroporated with Grg5 siRNAs.
(A) The right-hand half of the spinal cord is electroporated, as indicated
by GFP expression. (B) In situ hybridization of Grg5 mRNA showing
efficient ablation of Grg5 mRNA (asterisk) on the Grg5 siRNAelectroporated side. (C,D) Confocal micrographs of sectioned chick
spinal cords showing immunohistochemical analysis of motor column
markers. (E) Bar chart showing the percentage of motor columns in
electroporated Grg5 siRNAs (black bars), control siRNAs (gray bars) and
contralateral non-electroporated (white bars, set as 100%) sides of the
spinal cord at forelimb regions. n=4-5 embryos, mean ± s.e.m.
Grg5 and Nolz1 cooperate to induce Lim1
expression in ectopic MNs
At later stages of development, Nolz1 is localized to LMCl neurons,
suggesting potential roles for Nolz1 in regulating their development
(Fig. 2L,M). RA signaling pathways induce LMCl identity in
particular by upregulating Lim1, which directs the dorsal trajectories
of LMCl axons in the limb (Sockanathan and Jessell, 1998; 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 Islet2specific 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
Fig. 8. Coexpression of Nolz1 and Grg5 induces Hoxc6 expression
and partial lateral LMC specification. (A-C) Confocal micrographs of
ventral chick embryonic spinal cords showing that the onset of Nolz1
expression occurs prior to that of Hoxc6. The dashed line outlines the
spinal cord. (D-G) Confocal micrographs of electroporated dorsal right
halves of St 23 chick spinal cords. Grg5 expression is not shown but is
~95-98% coincident with exogenous Nolz1. The dashed line marks the
lateral boundary of the spinal cord. (H,I) Confocal micrographs of the
ventral MN domain of St 23/24 chick embryonic spinal cords
electroporated on the right. Yellow cells in panel I mark thoracic MNs
that express Hoxc6. (J-M) Confocal micrographs of electroporated dorsal
right halves of St 23 chick spinal cords. The dashed line marks the lateral
boundary of the spinal cord. The right-hand side is electroporated in all
cases. The high levels of Nolz1 elicited by overexpression preclude the
detection of endogenous Nolz1 under these imaging conditions.
(N) Model for Nolz1-dependent specification of MN subtype identity.
Orange lines mark cells expressing Nolz1. Broken orange line refers to
transient Nolz1 expression. Nolz1 downregulates Lim3 expression in
prospective Lim3-negative MNs and, in a complex with Grg5, maintains
the expression of key MN determinants. At limb levels, Nolz1-Grg5
complexes confer forelimb LMC identity through induction of Hoxc6,
and induce Lim1 expression in prospective LMCl MNs.
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.
DEVELOPMENT
through transcriptional repressor functions, and provide a
mechanism that links Hoxc6-dependent specification of forelimb
LMC identity with RA signaling pathways.
DISCUSSION
Here, we show that RA signals induce Nolz1 expression in the chick
ventral spinal cord, and that Nolz1 mediates novel and known RAdependent events that specify columnar and divisional identities in
postmitotic MNs (Fig. 8N). Our results suggest a model in which
paraxial mesoderm-derived RA signals induce Nolz1 in postmitotic
MNs, where it regulates the diversification and maintenance of
Lim3– MNs, prior to triggering forelimb LMC specification through
Hoxc6 induction. At limb levels, RA synthesized in paraxial
mesoderm and in MNs maintains Nolz1 expression, which acts to
upregulate Lim1 expression in late-born prospective LMCl neurons.
We discuss below how Nolz1 regulates the specification, function
and survival of postmitotic Lim3– MNs in the context of known
transcriptional hierarchies that orchestrate these events.
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 Nolz1Grg5 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 NkxGrg 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 Nolz1Grg5 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).
RESEARCH ARTICLE
239
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 limblevel 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 cellautonomously 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 noncell-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 Nolz1Grg5 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 Nolz1Grg5 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.
DEVELOPMENT
Nolz1 regulates motoneuron identity
RESEARCH ARTICLE
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.
We thank Shaneka Lawson for technical assistance; Jeremy Dasen, Tom Jessell,
Ben Novitch and Sam Pfaff for antibodies and reagents; Alex Kolodkin, Ben
Novitch and Ye Yan for comments on the manuscript; and members of the
Sockanathan laboratory for scientific discussions. Funding for this work was
provided by grants from NINDS (NIH), the Robert Packard Center for ALS
Research and the Wings over Wall Street Campaign by the Muscular Dystrophy
Association. Deposited in PMC for release after 12 months.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/2/231/DC1
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.
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.
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.
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.
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.
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals
and transcriptional codes. Nat. Rev. Genet. 1, 20-29.
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, 249261.
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.
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.
Maden, M. (2002). Retinoid signalling in the development of the central nervous
system. Nat. Rev. Neurosci. 3, 843-853.
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.
Development 136 (2)
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.
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.
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.
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.
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.
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.
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 MNdependent step in interneuron differentiation. Cell 84, 309-320.
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.
Prasad, A. and Hollyday, M. (1991). Development and migration of avian
sympathetic preganglionic neurons. J. Comp. Neurol. 307, 237-258.
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.
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.
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.
Sharma, K., Leonard, A. E., Lettieri, K. and Pfaff, S. L. (2000). Genetic and
epigenetic mechanisms contribute to MN pathfinding. Nature 406, 515-519.
Sockanathan, S. and Jessell, T. M. (1998). MN-derived retinoid signaling specifies
the subtype identity of spinal MNs. Cell 94, 503-514.
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.
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.
Tanabe, Y., William, C. and Jessell, T. M. (1998). Specification of MN identity by
the MNR2 homeodomain protein. Cell 95, 67-80.
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.
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 celltype-specific protein-protein interactions. Cell 110, 237-249.
Tosney, K. W., Hotary, K. B. and Lance-Jones, C. (1995). Specifying the target
identity of motoneurones. BioEssays 17, 379-382.
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.
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.
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.
DEVELOPMENT
240