3838 RESEARCH ARTICLE
Development 139, 3838-3848 (2012) doi:10.1242/dev.080994
© 2012. Published by The Company of Biologists Ltd
Zebrafish rest regulates developmental gene expression but
not neurogenesis
Fatma O. Kok1,2, Andrew Taibi1, Sarah J. Wanner3, Xiayang Xie4, Cara E. Moravec1,5, Crystal E. Love3,6,
Victoria E. Prince3,6, Jeff S. Mumm4 and Howard I. Sirotkin1,2,5,*
SUMMARY
The transcriptional repressor Rest (Nrsf) recruits chromatin-modifying complexes to RE1 ‘silencer elements’, which are associated
with hundreds of neural genes. However, the requirement for Rest-mediated transcriptional regulation of embryonic development
and cell fate is poorly understood. Conflicting views of the role of Rest in controlling cell fate have emerged from recent studies.
To address these controversies, we examined the developmental requirement for Rest in zebrafish using zinc-finger nucleasemediated gene targeting. We discovered that germ layer specification progresses normally in rest mutants despite derepression of
target genes during embryogenesis. This analysis provides the first evidence that maternal rest is essential for repression of target
genes during blastula stages. Surprisingly, neurogenesis proceeds largely normally in rest mutants, although abnormalities are
observed within the nervous system, including defects in oligodendrocyte precursor cell development and a partial loss of facial
branchiomotor neuron migration. Mutants progress normally through embryogenesis but many die as larvae (after 12 days).
However, some homozygotes reach adulthood and are viable. We utilized an RE1/NRSE transgenic reporter system to dynamically
monitor Rest activity. This analysis revealed that Rest is required to repress gene expression in mesodermal derivatives including
muscle and notochord, as well as within the nervous system. Finally, we demonstrated that Rest is required for long-term repression
of target genes in non-neural tissues in adult zebrafish. Our results point to a broad role for Rest in fine-tuning neural gene
expression, rather than as a widespread regulator of neurogenesis or cell fate.
INTRODUCTION
Context-dependent regulation of gene expression is key to the
generation of cell-type diversity during development. RE1silencing transcription factor [Rest; also known as Neuronrestrictive silencing factor (Nrsf)] is thought to play a central role
in the transcriptional repression necessary to achieve neuralspecific gene expression (Chong et al., 1995; Schoenherr and
Anderson, 1995). The ~23 bp repressor element 1/neuronrestrictive silencing element (RE1/NRSE) is the binding site for
Rest and is associated with hundreds of neural genes in mammals
(Bruce et al., 2004; Lunyak et al., 2002). Analysis of other
vertebrate genomes, including zebrafish, has revealed that many
possess rest homologs and comparable numbers of RE1 sites
(Mortazavi et al., 2006).
Rest alters chromatin structure by mediating the assembly of
multiple chromatin-modifying complexes. The N-terminal
repressor domain of Rest recruits Sin3, which dynamically interacts
with a wide range of repressor proteins including the methyl-DNAbinding protein MeCP2 and histone deacetylases (HDACs) 1/2
(Grzenda et al., 2009; Naruse et al., 1999). The Rest C-terminal
1
Department of Neurobiology and Behavior, Stony Brook University, Stony Brook,
NY 11794, USA. 2Graduate Program in Molecular and Cellular Biology, Stony Brook
University, Stony Brook, NY 11794, USA. 3Department of Organismal Biology and
Anatomy, University of Chicago, Chicago, IL 60637, USA. 4Department of Cellular
Biology and Anatomy, Georgia Health Sciences University, Augusta, GA 30912, USA.
5
Genetics Graduate Program, Stony Brook University, Stony Brook, NY 11794, USA.
6
Committee on Development, Regeneration and Stem Cell Biology, The University of
Chicago, Chicago, IL 60637, USA.
*Author for correspondence ([email protected])
Accepted 18 July 2012
domain interacts with CoREST (Ballas et al., 2001; Grimes et al.,
2000) to assemble complexes that include HDACs 1/2 and histone
H3 K9 methyltransferases (Lunyak et al., 2002; Roopra et al.,
2004; Shi et al., 2003). The full repertoire of genes under the
control of Rest is not regulated in unison and the context-specific
requirements for Rest-mediated repression are unknown.
Initially, the chief function of Rest was considered to be the
repression of neural genes in non-neural tissues. Recently, a more
complex understanding of Rest has emerged. Rest also modulates
neural gene expression within developing neurons and embryonic
stem (ES) cells (Ballas et al., 2005; Singh et al., 2008). In these
contexts, repression is transient, and Rest is proposed to delay
target gene expression until the proper time. Consistent with this
proposal, the transition from ES cell to neural progenitor to
differentiated neuron is accompanied by reduction in Rest levels
(Ballas et al., 2005; Guardavaccaro et al., 2008; Westbrook et al.,
2008). However, the function of Rest in cultured stem and
progenitor cells is controversial. Studies have reached opposing
conclusions on the requirement for Rest as a regulator of stem cell
pluripotency. Some have concluded that Rest is a key regulator of
pluripotency or of the emergence of the neural lineage from stem
cells (Singh et al., 2008; Sun et al., 2008; Gupta et al., 2009),
whereas other studies differ in this view (Jørgensen et al., 2009a;
Jørgensen et al., 2009b; Singh et al., 2008; Westbrook et al., 2008).
Because a variety of factors influence the ability of cells to divide
and differentiate, the outcomes of such experiments vary depending
on the cell line and culture conditions employed.
Rest loss-of-function analysis has yet to provide a clear view of
its role during embryogenesis. In the mouse Rest knockout,
widespread cell death accompanies lethality by E11.5 (Chen et al.,
1998). Expression of a few neural genes was observed in nonneural tissues, but the early lethality precluded further analysis;
DEVELOPMENT
KEY WORDS: Neurogenesis, REST (NRSF), RE1/NRSE
specific molecular and cellular defects underlying the lethality
remain unknown. In chick, inhibiting Rest with a dominantnegative construct resulted in ectopic expression of a few neural
genes in non-neural tissues, but not in alterations of fate (Chen et
al., 1998). Blocking Rest activity in Xenopus resulted in ectodermal
patterning defects and the derepression of target genes (Olguín et
al., 2006a). Previously, we knocked down zebrafish Rest using
morpholinos and observed alterations of Hedgehog signaling
(Gates et al., 2010).
To circumvent caveats associated with knockdowns and
dominant-negative constructs, we employed zinc-finger nucleases
to disrupt zebrafish Rest. Our in-depth analysis of the rest mutant
established that expression of RE1-containing genes is enhanced in
the mutant, and that blastula stage repression of some target genes
requires maternal rest. Unexpectedly, neurogenesis progresses
largely normally in rest mutants, although abnormalities were
observed in oligodendrocyte precursor cells and in facial
branchiomotor neuron migration. Analysis of an RE1/NRSE
transgenic reporter system revealed that Rest represses gene
expression in mesodermal derivatives including muscle and
notochord, as well as within the developing nervous system.
Although most rest homozygous zebrafish mutants die during
larval stages, some survive to maturity. In these fish, some RE1containing genes are inappropriately expressed in adult organs,
demonstrating a long-term role for Rest in the repression of target
genes in non-neural tissues. Together, these results establish Rest
as a repressor of gene expression during embryogenesis and in
adult non-neural tissue, but not as a broad regulator of cell fate or
differentiation.
MATERIALS AND METHODS
Zinc-finger nuclease gene targeting
ZiFiT (http://www.zincfingers.org/software-tools.htm) was used to identify
zinc-finger nuclease (ZFN) target sites that exclusively comprise GXX
sequences. Two zinc-finger arrays were designed against each ‘half-target’
site (supplementary material Table S1). Conventional cloning strategies
were employed to assemble arrays from clones in the Addgene ZF Kit.
Each ZF array was cloned into KpnI/BamHI sites of the FokI-RR/FokI-DD
vectors (Addgene). ZFN mRNA was synthesized using the mMESSAGE
mMACHINE Kit (Ambion) and 10-25 pg of mRNA was injected into
blastomeres of one-cell embryos. At 24 hours postfertilization (hpf),
embryos were screened by PCR using primers 181 (5⬘-CTGAGGGGAAGCAGATGATG-3⬘) and 184 (5⬘-TGTCCATGCTGTATCTCACGA-3⬘).
Quantitative PCR
Total RNA was extracted from tissues or pools of five embryos using
Trizol (Invitrogen). SuperScript II reverse transcriptase (Invitrogen) was
used to synthesize cDNA from 0.2-0.5 g of total RNA. Quantitative (q)
PCR was carried out with a LightCycler 480 (Roche) using 2⫻ FastStart
SYBR Green Master (Roche) or Quanta SYBR Green (Quanta
Biosciences). Total RNA from each sample was normalized to -actin. In
each experiment, three pools of three to five embryos or one adult organ
were run in duplicate. Primer pairs are listed in supplementary material
Table S3.
TSA treatments
Pronased embryos were placed in 40 nM Trichostatin A (TSA) or DMSO
at the eight-cell stage and collected at 4 hpf for mRNA isolation.
Whole-mount in situ hybridization and immunostaining
Whole-mount in situ hybridization was performed as described by Thisse
et al. (Thisse et al., 1993). Rest immunostaining was performed as
described (Mapp et al., 2011).
RESEARCH ARTICLE 3839
Fig. 1. Zinc-finger nuclease-mediated targeting of zebrafish rest.
(A)Sequence chromatograms from wild-type siblings (left) and rest
mutants (right). The 7 bp deletion (7) in SBU29 and 4 bp insertion (+4)
in SBU34 are illustrated beneath. (B)Domain structure of wild-type Rest
and truncated proteins produced by SBU29 and SBU34 mutations.
(C)Animal pole views of 6-hpf wild-type and MZrestsbu29/sbu29 embryos.
Rest immunoreactivity, TOPRO-3 staining of nuclei, and merged
channels are shown. Abundant cytoplasmic Rest protein is detected in
wild-type embryos, but not in MZrestsbu29/sbu29 mutants. Confocal
images are single 1-m stacks taken at 40⫻ magnification.
RESULTS
Targeted rest disruption with zinc-finger
nucleases
We disrupted zebrafish rest (Fig. 1; supplementary material Tables
S1, S2) using zinc-finger nucleases (ZFNs) (Doyon et al., 2008; Kim
et al., 1996; Meng et al., 2008) and recovered ten mutations in the
first exon of rest. The majority of our studies utilized the restsbu29
allele (a 7 bp deletion), but we also examined viability of the restsbu34
allele (a 4 bp insertion). Both mutations produce similar frameshifts
upstream of the DNA-binding domain and are likely null alleles.
Unlike the mouse Rest knockout, restsbu29/sbu29 and restsbu34/sbu34
embryos appear grossly normal throughout embryogenesis and early
larval stages [10 days postfertilization (dpf)]. However, adult mutants
were not recovered at the expected Mendelian ratios. Of adult
offspring of restsbu29/+ intercrosses, only 17/214 (7.9%) were
homozygous mutants. Likewise, only 3/237 (1.3%) of adult offspring
from restsbu34/+ intercrosses were homozygous mutants. A subset of
the restsbu29/sbu29 adults had posterior defects, while all three of the
restsbu34/sbu34 mutants were scrawny, had a severely curved axis and
died by 3 months. Some surviving restsbu29/sbu29 fish were fertile.
Although we cannot exclude the possibility that the Restsbu34 protein
has antimorphic activity, it is likely that the phenotypic variations
stem from genetic background variations within the wild-type strain
that was used for targeting. The surviving mutants provided a unique
opportunity to study the requirement for Rest in larvae and adults.
DEVELOPMENT
Zebrafish rest knockout
3840 RESEARCH ARTICLE
Development 139 (20)
RT-PCR analysis of restsbu29/sbu29 mutants revealed that both
wild-type and restsbu29 messages are present at 6 hpf (early
gastrula), but not at later stages (data not shown). The presence of
wild-type rest mRNA in restsbu29/sbu29 gastrulae reflects maternal
rest expression and is consistent with our previous analysis (Gates
et al., 2010). The lack of morphological defects in rest mutant
embryos might be due to compensation by maternal rest mRNA.
To eliminate maternal Rest activity, we generated embryos devoid
of both maternal and zygotic rest (MZrestsbu29/sbu29) by crossing
homozygous restsbu29/sbu29 females and males. To our surprise, these
mutants also lacked overt morphological defects, demonstrating
that Rest is dispensable for embryogenesis in zebrafish.
The nature of the rest lesions (indels) and their position within
the first exon make it unlikely that either readthrough or alternative
splicing can produce functional protein, as the first exon encodes
zinc fingers that constitute part of the DNA-binding domain. To
confirm the absence of Rest protein, we performed
immunohistochemistry, which revealed abundant Rest protein in
wild-type 6-hpf embryos but not in MZrestsbu29/sbu29 mutants (Fig.
1C). This supports the conclusion that SBU29 is a null allele.
Rest represses target gene expression during
embryogenesis
The zebrafish genome has ~1000 canonical RE1 sites (Mortazavi
et al., 2006) (our unpublished results) and mammalian Rest binds
teleost RE1 elements (Johnson et al., 2009; Tan et al., 2010). To
determine whether loss of Rest affects target gene expression, we
selected a set of RE1-containing genes with diverse cellular
functions to analyze in the mutant. We studied their expression by
qRT-PCR in MZrestsbu29/sbu29 and wild-type embryos between 4 hpf
(late blastula) and 16 hpf (mid-somitogenesis) (Fig. 2). During this
time, expression of some Rest target genes was enhanced in
mutants. At 4 hpf, when the embryo comprises pluripotent
blastomeres, the expression levels of most RE1-containing genes
are either very low or absent in wild-type embryos (Fig. 2). At this
stage, transcripts of gpr27, kcnh8 and snap25b can be detected in
MZrestsbu29/sbu29 mutants, but not in controls (Fig. 2A-C). Similarly,
during somitogenesis (11 and 16 hpf), expression of these genes is
low or absent in wild-type embryos, but much more robust in the
mutant. Injection of morpholinos to impede rest translation also
enhanced snap25b expression (data not shown).
Expression of bdnf, snap25a, grin1a, pcad (pcdh1g2) and
cacng2 was enhanced in a stage-dependent manner in
MZrestsbu29/sbu29. However, we identified a group of Rest target
genes (neurod, spop, gfap and bsx) that showed unchanged
expression levels in MZrestsbu29/sbu29 mutants, suggesting that both
genomic context and cell type influence Rest activity. Consistent
with our findings, previous studies have shown that Rest function
is cell type dependent, and that blocking Rest does not derepress
all target genes (Belyaev et al., 2004; Chen et al., 1998; Otto et al.,
2007). Our findings provide the first evidence that Rest is required
to suppress target gene expression in blastulae.
DEVELOPMENT
Fig. 2. Expression levels of RE1-containing genes during early development of rest mutants. qPCR analysis showing fold differences
relative to MZrestsbu29/sbu29 transcript levels (defined as 1). (A-C)gpr27, kcnh8 and snap25b are either not expressed or expressed at low levels in
wild-type zebrafish embryos, but are expressed in MZrestsbu29/sbu29 mutants. (D-F)bdnf, snap25a and grin1a show stage-specific alterations in
expression in rest mutants. (G-L)Expression of pcad, cacng2, neurod, gfap, spop and bsx is not significantly altered in the rest mutant. Error bars
indicate s.e. ND*, not detected.
Fig. 3. Maternal rest suppresses target genes in mid-blastula
embryos. qPCR analysis of (A) snap25b, (B) bdnf, (C) pcad and
(D) gpr27 expression in mid-blastula zebrafish embryos. Comparison of
transcript levels in wild-type, Zrestsbu29/+ and Mrestsbu29/+ embryos
reveals derepression of target genes in Mrest but not Zrest embryos.
Fold comparisons are relative to Mrest transcript levels (defined as 1).
Maternal (M) and zygotic (Z) embryos were obtained by crossing female
and male restsbu29/sbu29 mutants, respectively, to wild types. Error bars
indicate s.e. ND*, not determined.
Maternal Rest function is required for early gene
regulation
Our initial experiments did not distinguish the contributions of
zygotic versus maternal Rest to the repression of target genes. To
elucidate the role of maternal Rest in the regulation of RE1containing target genes, we compared gene expression in progeny
from restsbu29/sbu29 females and wild-type males (maternal,
Mrestsbu29/+ embryos) to offspring of restsbu29/sbu29 males and wildtype females (zygotic, Zrestsbu29/+ embryos) as well as wild-type
controls. Although Zrestsbu29/+ fish have the same genotype as
Mrestsbu29/+ fish, early Rest activity stems from maternal rest
mRNA that is absent from Mrestsbu29/+ embryos.
Four RE1-containing genes that are expressed at blastula stages
were used for this analysis. qRT-PCR demonstrated that loss of
maternal rest function increased levels of snap25b, bdnf and pcad
at mid-blastula stage (Fig. 3A-C). In the reciprocal experiment, the
expression levels of the RE1-containing target genes in Zrestsbu29/+
embryos were more similar to wild-type levels (Fig. 3A-C),
suggesting that maternal Rest function is both necessary and
sufficient for early repression of RE1-containing genes. gpr27
expression was undetectable in all three genotypes (Fig. 3D),
indicating that either maternal or zygotic rest is sufficient for
repression of this locus. These findings provide the first evidence
that maternal rest suppresses zygotic gene expression.
REST-mediated histone modifications in blastulae
are target gene dependent
Rest recruits chromatin-modifying enzymes to RE1/NRSE sites to
alter histone acetylation and methylation states (Ballas et al., 2001;
Huang et al., 1999). To determine whether effects on histone
acetylation are central to the function of Rest during blastula stages,
we examined the effects of the HDAC inhibitor TSA on target gene
expression in wild-type and MZrestsbu29/sbu29 embryos (Fig. 4). The
difference between the effects of TSA on wild type versus rest
mutants reflects the relative contribution of HDAC-independent
activities of Rest, namely histone methylation. At least three
response profiles were apparent among the Rest target genes
analyzed.
RESEARCH ARTICLE 3841
Fig. 4. Histone deacetylase contribution to Rest-mediated
inhibition of target genes is locus specific. qPCR analysis of RE1containing gene expression at mid-blastula stage in wild-type and
MZrestsbu29/sbu29 zebrafish embryos treated with DMSO or the HDAC
inhibitor TSA. The fold differences are relative to transcript levels in
MZrestsbu29/sbu29 DMSO (defined as 1). (A,B)TSA treatment enhances
expression of snap25a and gpr27 in both MZrest and wild-type
embryos compared with DMSO controls. (C)TSA treatment of wild-type
embryos produces a much smaller increase in snap25b expression than
does removing Rest function. This suggests that Rest-dependent
histone methylation is central to repression of snap25b expression.
(D-F)HDAC inhibition has little effect on pcad, spop and grin1a
expression. Error bars indicate s.e.
The expression levels of snap25a and gpr27 in TSA-treated
wild-type embryos were comparable to DMSO-treated
MZrestsbu29/sbu29 embryos (Fig. 4A,B). Alone, these findings
suggest that HDAC recruitment might be the major component of
Rest function at these loci. However, examination of transcript
levels in TSA-treated MZrestsbu29/sbu29 mutants revealed more
intricate effects of Rest on chromatin. At both loci the impact of
HDAC inhibition was greater in the absence of Rest than in wildtype embryos. This implies that Rest-mediated effects on histone
methylation act cooperatively with HDACs recruited by other
transcriptional regulators to repress transcription of these loci.
snap25b expression showed a very different response to TSA.
Wild-type TSA-treated embryos showed a 3-fold increase in
snap25b levels compared with DMSO-treated controls (Fig. 4C).
In comparison, DMSO-treated rest mutants and TSA-treated rest
mutants showed similar ~57-fold increases relative to wild-type
snap25b levels. These results show that, whereas blocking histone
deacetylation minimally enhances the expression of snap25b in
wild-type embryos at mid-blastula stage, the effects of Rest
inhibition are far greater. This demonstrates that HDACindependent Rest modifications play a major role at the snap25b
locus in blastulae.
pcad, spop and grin1a were also challenged with TSA, but their
expression profiles were not statistically different in wild type and
rest mutants (Fig. 4D-F). Although we cannot distinguish direct
from indirect actions of HDAC inhibition, taken together these
DEVELOPMENT
Zebrafish rest knockout
3842 RESEARCH ARTICLE
Development 139 (20)
observations reveal complex regulatory interactions at Rest target
genes with histone deacetylation playing dramatically different
roles at different loci. Surprisingly, the two zebrafish snap25
orthologs respond differently to TSA treatment, with repression of
snap25b being almost entirely independent of HDAC activity.
We next asked whether key Rest co-factors were expressed in
zebrafish blastulae. qRT-PCR analysis revealed that rcor1 (Corest1),
rcor2 (Corest2), sin3aa, sin3ab and sin3b transcripts are present at
blastula stage (supplementary material Fig. S1). Of these markers,
only the expression of rcor1 was enhanced in MZrestsbu29/sbu29
mutants (supplementary material Fig. S1). We analyzed rcor1
expression by RNA in situ hybridization and observed that many
domains of expression overlap with rest expression (data not shown)
and that rcor1 expression is slightly more robust in the mutant.
Fig. 5. Neurogenesis progresses normally in rest mutants. Wholemount in situ hybridization for the pan-neural marker huC in stagematched (A,B) 2-somite, (C,D) 8-somite, (E,F) 14-somite and (G,H) 24hpf wild type or rest heterozygotes and MZrestsbu29/sbu29 mutants. All
views are dorsal, anterior to the left. MZrest mutants did not show
ectopic or precocious huC expression. To control for subtle differences
in developmental timing and genetic background, comparisons were
undertaken in crosses of restsbu29/sbu29 females to restsbu29/+ males and
between wild-type intercrosses and MZrestsbu29/sbu29 intercrosses.
Genotypes were determined by PCR.
Neurogenesis progresses normally in rest mutants
Rudimentary observation of rest mutant locomotor behavior and
gross neural anatomy did not reveal abnormalities. To further
investigate the requirement for Rest in neurogenesis, the domains
of proneural and pan-neural markers were assayed in
MZrestsbu29/sbu29 mutants by RNA in situ hybridization.
Examination of the proneural markers ascl1a and neurog1 did not
reveal abnormalities in MZrestsbu29/sbu29 mutants (supplementary
material Fig. S3). If Rest modulates the differentiation of neural
progenitors, loss of Rest function might result in ectopic or
premature neural differentiation. We examined expression of the
pan-neural marker huC (elavl3) in MZrestsbu29/sbu29 mutants
between 10.5 hpf (2 somites) and 24 hpf. To our surprise, we did
not detect inappropriate huC expression in MZrestsbu29/sbu29 mutants
(Fig. 5). We also examined the expression of an huC:GFP
transgenic reporter in the MZrestsbu29/sbu29 mutants between 1 dpf
and 5 dpf, but did not observe alterations in expression (data not
shown). From these results, we conclude that Rest does not play a
fundamental role in zebrafish neurogenesis.
olig2:GFP was not altered in rest mutants, but a deficit was apparent
in the number of OPCs in the rest mutants at 50 hpf. On average, the
rest mutants had only 13.4±2.4 OPCs (± s.e.; n16) compared with
37.5±7.0 (n8) OPCs in sibling controls. OPC deficiencies remain
at 74 hpf (data not shown), but by 98 hpf the number of OPCs in the
mutants had recovered to levels comparable to sibling controls (Fig.
6). Similar deficits were observed in OPCs following Rest
knockdown using morpholinos (data not shown).
As in other species, the zebrafish OPC population is tightly
regulated and compensatory mechanisms can overcome deficits
(Kirby et al., 2006). The zebrafish rest mutant phenotype resembles
the mouse Olig1 knockout, in which early deficits of OPCs are
subsequently overcome (Lu et al., 2002). Recent studies have
provided evidence that Rest regulates glial differentiation,
including emergence of oligodendrocytes (Abrajano et al., 2009;
DeWald et al., 2011; Soldati et al., 2012). Our in vivo results
implicate Rest-regulated gene expression in the development of the
OPC lineage in zebrafish.
olig2+ oligodendrocyte precursors are reduced in
rest mutants
Neural fate determination appears grossly normal in rest mutants. To
study subtle differences in neural subpopulations that lack functional
Rest, we examined olig2+ cells in MZrestsbu29/sbu29 using an
olig2:GFP transgene (Fig. 6). This transgenic line marks progenitors
in the ventral spinal cord domain that give rise to motor neurons and
oligodendrocyte precursor cells (OPCs). By 3 dpf, OPCs occupy
distinct positions after migrating dorsally from the progenitor
population (Park et al., 2002). The ventral spinal cord expression of
Facial branchiomotor neuron migration is
impaired in rest mutants
Facial branchiomotor neurons (FBMNs) are a subset of the cranial
branchiomotor neurons common to all vertebrates. Zebrafish
FBMNs are born in rhombomere (r) 4 of the hindbrain and migrate
tangentially to r6-r7 (Chandrasekhar, 2004). Migration begins after
the first neurons are born at 16 hpf and is complete by 48 hpf,
when the FBMNs have reached their final destination in r6-r7. Rest
is expressed in FBMNs (supplementary material Fig. S4) and
inhibition of Rest function by morpholino knockdown or a
DEVELOPMENT
Rest is not required for germ layer formation in
zebrafish
Studies from other organisms have not yielded a clear picture of
the role for Rest during early development. In Rest knockout mice,
midbrain head mesenchyme and myotomal cells in the somite are
disorganized (Chen et al., 1998). However, overall germ layer
formation and early patterning seem to be minimally affected
(Chen et al., 1998). By contrast, perturbing Xenopus Rest expands
the neural plate and reduces neural crest (Olguín et al., 2006b). To
study whether Rest function is necessary for germ layer
specification in zebrafish, we analyzed the expression of early
patterning genes in MZrestsbu29/sbu29 mutants. During gastrulation,
expression of mesodermal (chd, ntl, myod1, foxa2), endodermal
(foxa2) and neural (pax3) markers was not altered in
MZrestsbu29/sbu29 mutants (supplementary material Fig. S2). These
results suggest that germ layer specification does not require Rest.
Zebrafish rest knockout
RESEARCH ARTICLE 3843
Fig. 6. Rest function is crucial for the regulation of
oligodendrocyte precursor cells in the dorsal spinal cord and for
facial branchiomotor migration. (A-D)Lateral views of live 50-hpf
and 98-hpf Tg(olig2:GFP) (A,C) and Tg(olig2:GFP); MZrestsbu29/sbu29
mutants (B,D). The number of migrating olig2+ oligodendrocyte
precursors (arrows) is significantly reduced in rest mutants at 50 hpf (B)
compared with controls (A). The number of migrating OPCs (arrows) in
98-hpf MZrestsbu29/sbu29 mutants (D) is similar to controls (C).
(E-G)Dorsal views of the hindbrain of 48-hpf Tg(islet1:GFP) transgenic
zebrafish embryos. Representative embryos from a restsbu29/+, restsbu29/+;
islet1:GPF intercross. (E)rest+/+ embryo showing normal FBMN
migration into r6-r7. (F)FBMNs in restsbu29/+ embryos have only subtle
migration defects (arrows). (G)In restsbu29/sbu29 embryos, a significant
proportion of FBMNs remain in r4-r5 (bracket).
dominant-negative approach was previously shown to cause defects
in FBMN migration (Mapp et al., 2011). Using an islet1:GFP
transgene, we examined rest mutants for defects in FBMN
migration (Fig. 6). Following an intercross of restsbu29/+ and
restsbu29/+; islet1-GFP fish, we observed three phenotypic classes.
Out of 59 embryos, ten had wild-type FBMN positioning (Fig. 6E).
Genotype analysis revealed that nine of these were wild type for
rest and one was a heterozygote. A second class showed very subtle
changes in FBMN migration, exhibiting minor disorganization of
neurons in r6 and r7 and a slight increase in the number of FBMNs
remaining in r5 at 48 hpf (Fig. 6F). All 40 embryos in this class
were restsbu29/+ heterozygotes. The final phenotypic class showed
more severe migration defects and seven out of nine of these
embryos were restsbu29/sbu29 homozygous mutants. In the mutants,
many neurons were unable to migrate properly into r6, remaining
instead in the r4-r5 region (Fig. 6G). These phenotypes closely
resemble previously described Rest morphant phenotypes, and
confirm a requirement for Rest function in control of FBMN
migration.
Repression of RE1-containing genes by Rest at
late embryonic and larval stages
Having observed derepression of Rest target genes in the mutant
prior to 13 hpf (Fig. 2), we sought to establish whether Rest
continues to repress target genes at later stages. As the embryo
begins to mature and neurons differentiate, the overall expression
levels of Rest target genes increase in wild-type embryos. We
compared expression levels of RE1-containing genes in
Monitoring Rest activity with a transgenic
reporter system
Although Rest-mediated repression of target genes in vitro is well
studied, little is known about the spatial or temporal requirements
for Rest in intact animals. Analysis is complicated by the nature of
Rest activity: epigenetic chromatin modifications mediated by Rest
may endure once Rest protein is degraded. To dynamically map
tissues in the developing embryo where Rest activity is required,
we crossed the restsbu29 allele into four transgenic enhancer trap
lines [Et(NRSE-cfos:KalTA4), hereafter NRCK (X.X. and J.S.M,
unpublished)] that each contain an RE1/NRSE element adjacent to
a minimal c-fos promoter upstream of KalTA4, an optimized Gal4VP16 driver (Distel et al., 2009).
Activity of these lines was monitored by crossing them to a
14xUAS:NfsB-mCherry transgenic reporter line that expresses a
fusion protein between nitroreductase (Ntr) and a monomeric red
fluorescent reporter, mCherry [Tg(UAS-E1b:NfsB-mCherry)c264,
hereafter 14xNtrCh] (Davison et al., 2007). The UAS-linked
mCherry reporter is dependent on KalTA4 activation. Therefore,
the spatial and temporal expression of mCherry depends on the
genomic context of the KalTA4 enhancer trap insertions. Owing to
the influence of the RE1/NRSE element, NRCK lines express
KalTA4 primarily in the nervous system, which in turn restricts
expression of mCherry to the nervous system in double transgenics.
Two of the lines, NRCKgmc606 and NRCKgmc607, drive expression
principally in the nervous system and weakly in the mesoderm.
Two additional lines, NRCKgmc632 and NRCKgmc641, display
expression patterns that are almost exclusively restricted to the
nervous system. In all cases, expression of mCherry reporters was
altered in rest mutants.
The first line, NRCKgmc606, drives mCherry expression in the
spinal cord, a few notochord cells and in an occasional muscle fiber
(Fig. 7A). The progeny from crosses between restsbu29/+;
NRCKgmc606; 14xNtrChc264/+ and restsbu29/+ fish fell into two
phenotypic classes. The first class expressed mCherry in the
expected predominately neural NRCKgmc606 pattern (n33/50) and
comprised only wild-type and rest heterozygous embryos (Fig.
7A). By contrast, the remaining embryos (n17/50) had
significantly expanded mCherry expression, with many notochord
cells and numerous muscle fibers expressing mCherry, and were all
rest mutants (class 2, Fig. 7B).
The NRCKgmc607 line expresses mCherry within the spinal cord,
but also in a few muscle fibers and enteric neurons (Fig. 7C).
Among progeny of restsbu29/+; NRCKgmc607; 14xNtrChc264/+ and
restsbu29/+ crosses, a phenotype (class 2) emerged that showed
expanded neural expression, widespread expression in muscle of
the trunk and head and in notochord (Fig. 7D). Genotyping
revealed that 11/13 (84.6%) class 2 embryos were rest mutants
(Fig. 7J). Conversely, only 4/51 (7.8%) of class 1 embryos, which
displayed the expected expression pattern, were rest mutants. In
summary, analysis of the NRCKgmc606 and NRCKgmc607 lines
DEVELOPMENT
MZrestsbu29/sbu29 mutants and wild types by qRT-PCR between 1
and 19 dpf. Expression levels of nearly all target genes were
comparable between mutants and wild types (supplementary
material Fig. S5). Notable exceptions were gpr27, which showed
elevated expression in rest mutants at all stages, and kcnh8, bdnf
and pcad, which were elevated at 24 hpf in the mutants. We also
employed more sensitive approaches to assay snap25a expression.
However, neither in situ hybridization nor a new snap25a-GFP
transgenic reporter line (data not shown) revealed evidence of
ectopic expression.
3844 RESEARCH ARTICLE
Development 139 (20)
Fig. 7. RE1/NRSE reporter transgenic lines
are ectopically expressed in rest mutants.
(A-H⬘) Lateral (A-F) or dorsal (G,H) views of live
RE1/NRSE transgenic reporter lines in wild-type
(A,C,E,G) or restsbu29/sbu29 (B,D,F,H) backgrounds.
Expression of each reporter was altered in the
mutants. The boxed region in G and H is shown
at higher magnification in G⬘ and H⬘. Arrows
mark midline cells, which are not as numerous in
the wild-type background. (I-L)The proportion of
embryos in each phenotypic and genotypic class
for each reporter. gmc606 and gmc641 larvae
are 3 dpf; gmc607, 6 dpf; gmc632, 4 dpf.
The NRCKgmc641 results suggest that either mutant neurons
prematurely express the RE1/NRSE transgene or that newly
differentiated mutant neurons fail to migrate properly. By 5 dpf we
could no longer distinguish between class 1 and class 2 specimens,
as both had many mCherry-positive midline cells (data not shown).
Although we did not observe alterations in the subset of Rest
target genes that we studied at larval stages, analysis of the reporter
lines verified that Rest suppresses the expression of target genes
between 3 and 6 dpf in muscle and notochord. In addition, analysis
of these lines demonstrated that Rest is also required within the
developing nervous system to repress gene expression. Taken
together, these data suggest that Rest-mediated repression is highly
context dependent.
Rest is required to maintain the repression of
target genes in adult tissues
To determine whether Rest mediates long-term repression, we
isolated brain, muscle, liver, heart, spleen and pancreas from adult
wild-type and restsbu29/sbu29 fish and compared snap25b, grp27,
grin1a, bdnf and neurod transcript levels (Table 1). In wild-type
animals, each of these markers showed variable expression
between animals. These differences might stem from unique
environmental challenges faced by each animal over many months.
Although this variability might have obscured some effects, clear
trends emerged nonetheless. In rest mutants, expression of snap25b
was derepressed in pancreas, spleen and liver, whereas grin1a was
derepressed in pancreas and muscle (Table 1). Expression of bdnf
was enhanced in rest mutant spleens, but changes in neurod
expression were not apparent in any of the organs tested. It has
been suggested that Rest functions as a transcriptional activator in
DEVELOPMENT
demonstrates that Rest acts on RE1/NRSE elements to repress
expression of neighboring genes in mesodermal derivatives
including muscle and notochord between 3 and 6 dpf. To our
knowledge, this is the first evidence for Rest activity in the
notochord.
Two additional lines, NRCKgmc632 and NRCKgmc641, are
expressed almost exclusively within the nervous system. The
NRCKgmc632 line labels sparse cells within the spinal cord and
brain. At 4 dpf, the progeny of restsbu29/+; NRCKgmc632/+;
14xNtrChc264/+ and restsbu29/+ crosses again fell into two phenotypic
classes (Fig. 7E,F). The majority of embryos (65/85) displayed the
expected pattern of expression (class 1). The second class had
broader expression within the spinal cord, including numerous
interneurons (Fig. 7F). All 23 class 2 embryos that had broader
expression were rest mutants (Fig. 7K). Class 1 comprised 64 wildtype and heterozygous embryos and a single rest mutant.
The neural-specific NRCKgmc641 line expresses primarily in
secondary motor neurons. The progeny of restsbu29/+;
NRCKgmc641/+; 14xNtrChc264/+ and restsbu29/+ crosses fell into three
phenotypic classes at 3 dpf. In class 1, mCherry expression was
observed in two parallel rows of cells along the spinal cord (Fig.
7G,G⬘). Class 2 (n33) had similar expression except that
mCherry-positive cells were frequently observed in the midline
(Fig. 7H,H⬘). Genotypic analysis of class 2 revealed that mutants
were predominant, representing 66.7% (22 embryos), whereas only
3% (1 embryo) was wild type (Fig. 7L). Conversely, mutants
comprised only 9.2% (6/65 embryos) of class 1 (Fig. 7L). In class
3 (n31), fewer cells expressed mCherry and it was not possible to
assess the midline of these individuals. Genotype analysis of this
class revealed no enrichments of any genotype.
Zebrafish rest knockout
RESEARCH ARTICLE 3845
Marker
Muscle
Pancreas
Heart
Brain
Spleen
Liver
gpr27
snap25b
bdnf
grin1a
neurod
+
+++
+
+++
–––
++
+++
–––
++
–––
n.t.
–––
–––
–––
–––
+
–––
–––
–––
–––
+++
+
++
–––
–––
++
n.a.
–––
–––
–––
All markers were run on three independent tissue samples. Plus signs indicate the
number of samples in which the given marker was derepressed in the mutant.
n.t., not tested; n.a., no amplification.
adult brain (Kuwabara et al., 2004), but no significant alterations
in expression were observed in the targets that we analyzed from
this organ.
Although some restsbu29/sbu29 adults produce viable offspring,
their fertility and fecundity often wane more rapidly than those
of wild-type fish. We therefore examined the expression of RE1
target genes in ovaries and testes (Fig. 8). Strikingly, snap25b
expression was near the threshold of detection in wild-type
ovaries, but expression was more abundant in rest mutant
ovaries. In addition, levels of gpr27, kcnh8 and pcad expression
were elevated in the mutant ovaries. To determine whether this
effect was specific to female reproductive tissue, we examined
all the markers in testes, but only kcnh8 expression was
enhanced (Fig. 8). These observations demonstrate a longpostulated, but previously largely unproven, role for Rest in
long-term repression of RE1-containing genes in non-neural
tissue, and also reveal a high degree of tissue and locus
specificity in the effects of Rest on target genes.
DISCUSSION
To examine the requirement for Rest in the context of an intact
animal, we disrupted zebrafish rest using ZFNs. Based on
previously proposed roles for Rest, we anticipated that this
would cause inappropriate or failed neurogenesis. Contrary to
this expectation, the mutants underwent relatively normal
neurogenesis, although defects were observed in OPCs and in the
migration of the FBMNs (Fig. 6). We observed inappropriate
expression of genes and reporters in the mutant (Figs 2, 3, 6 and
7), but no clear alterations of cell fate (Fig. 5; supplementary
material Figs S2, S3).
Several in vitro studies have reported conflicting observations
regarding the role of Rest in stem cell maintenance during
development. Some reports assert that Rest is required for selfrenewal and pluripotency of ES cells (Gupta et al., 2009; Singh et
al., 2008), whereas others strongly contest this observation
(Buckley et al., 2009; Jørgensen et al., 2009a; Jørgensen et al.,
2009b; Yamada et al., 2010). Our results demonstrate that Rest is
not required to maintain pluripotency or self-renewal of developing
zebrafish blastomeres as germ layer specification proceeds
normally in rest mutants (supplementary material Fig. S2).
Although our findings do not reveal any requirement for Rest in
early embryos, paradoxically, some of the largest changes in gene
expression that result from Rest disruption occur during blastula
stages (Fig. 2). Under some conditions, this failure to restrain
inappropriate transcription might influence the proliferation or
developmental potential of these cells.
Does Rest function differ in zebrafish and mouse?
Mouse Rest mutants undergo widespread cell death and die by
E11.5 (Chen et al., 1998). By contrast, zebrafish rest mutants
survive for at least 2 weeks. The disparity between the zebrafish
and mouse phenotypes might stem from distinctive compensatory
mechanisms present in zebrafish, or from alterations in rest
function or targets between species. It is plausible that mechanisms
compensate for the lack of Rest function in zebrafish and that these
are less prominent in mammals. The most likely of such
mechanisms would involve a second rest or rest-like gene, as gene
duplicates are common in zebrafish (Postlethwait et al., 2000).
Functional similarities have been proposed between rest and
several invertebrate genes including spr-3, spr-4, charlatan and
tramtrack (Dallman et al., 2004; Lakowski et al., 2003; Tsuda et
al., 2006). A zebrafish homolog of one of these factors might also
compensate for loss of Rest function. We extensively searched the
zebrafish genome for rest-related genes and have not detected a
second rest ortholog or another rest-like gene. In addition, we
found that snap25a promoter-GFP transgenic lines that contain a
scrambled RE1 element generally mirror expression of the wildFig. 8. Rest target genes are
depressed in germ tissue of adult
rest mutants. qPCR analysis of RE1containing gene expression in wildtype and restsbu29/sbu29 mutant ovary
and testes (three examples of each
are shown). All fold differences are
relative to the average of the
transcript levels in the three mutants
(defined as 1). (A-D)Levels of
snap25b, kcnh8, gpr27 and pcad are
enhanced in the rest mutant ovary.
(E)Only kcnh8 expression was
enhanced in rest mutant testes.
DEVELOPMENT
Table 1. Derepression of RE1-containing genes in adult tissues
of rest mutants
type promoter (data not shown). If additional factors act on the
snap25a RE1 site, mutating the site should have caused
inappropriate expression of the reporter.
We favor the model that the phenotypic differences in mouse
and zebrafish rest mutations stem from differences in target
genes. Zebrafish have a comparable number of RE1 sites to other
vertebrates (Mortazavi et al., 2006). Fish RE1/NRSE elements
have been shown to bind mammalian Rest (Johnson et al., 2009)
and the mammalian and zebrafish Rest DNA-binding domains
exhibit a high degree of conservation (Gates et al., 2010).
However, a key difference might be in the positioning of
RE1/NRSE sites. Many genes have RE1/NRSE sites that are
conserved between fish and mammals, but some sites are unique.
Because regulatory elements are thought to be prime targets for
evolutionary change (King and Wilson, 1975), such differences
are expected. The cause of the lethality of the mouse Rest
knockout remains unknown, but it precedes neurogenesis. The
rampant cell death that is observed in the mouse knockout could
potentially result from misregulation of a single locus that is not
under the control of Rest in zebrafish.
While this manuscript was in preparation, a report was
published showing that a neural-specific conditional Rest
knockout does not alter neurogenesis, although effects were
noted in cultured cells isolated from mouse (Aoki et al., 2012).
Although further analysis of rest conditional knockouts is
required, these findings support our conclusion that Rest is not
essential for neurogenesis and highlight similarities between
Rest function in zebrafish and mice.
What is the role of Rest?
Given the size and frequency of RE1 sites, strong selective pressure
must maintain their presence throughout the zebrafish genome.
Although we identified embryonic requirements for Rest in OPC
development and in cranial nerve migration, these deficits are less
severe than would be expected for a master regulator of
neurogenesis. We also detected changes in the expression of RE1containing genes during embryogenesis and early larval
development. These changes were most apparent during blastula
and somite stages and largely reflected precocious gene expression.
The large fold differences between rest mutants and wild type
during these stages reflects low or absent expression in wild type
and more easily detectable expression in the mutant. Our data
provide the first in vivo evidence that Rest regulates gene
expression in ES cells (blastomeres). However, no alterations in
cell fate were observed in the mutants. Future analysis of the
mutants might reveal biases in the developmental potential of Restdeficient blastomeres, but our analysis does not support a broad
role for Rest in cell fate determination. In this context, Rest might
promote the fitness of stem cells by suppressing the premature
production of target transcripts.
During later stages of embryogenesis, many of the loci we
analyzed appeared to be largely unaffected by loss of Rest. Our
analysis of target gene expression during late embryonic and larval
stages relied primarily on qPCR, and subtle alterations in gene
expression might have gone undetected. However, we also
examined expression of a snap25a-GFP reporter line in the mutant
and failed to detect changes in expression in the absence of Rest at
these stages (data not shown). Simpler reporter constructs (the
NRSE KalTA4 enhancer trap lines) revealed substantial Rest
activity in mesodermal derivatives including muscle and notochord,
as well as within the nervous system (Fig. 7). These findings
suggest that Rest-mediated repression of RE1/NRSE sites is highly
Development 139 (20)
context dependent and such activity might be more apparent in the
milieu of the NRSE-basal promoter constructs. From these
observations, we suspect that the chief function of Rest at these
stages is to fine-tune the expression of RE1-containing genes rather
than as a robust silencer of expression.
Analysis of adult tissues from surviving rest mutants provided
the first proof that Rest is required in non-neural tissues for longterm suppression of target gene expression (Fig. 8, Table 1). These
effects were most pronounced in the germ tissue. Our anecdotal
observations suggest that rest mutants tend to have reduced fertility
compared with wild types. These observations suggest that Rest
might have a role in the maintenance of the adult germline or
simply contributes to overall fitness.
Rest has been proposed to function within the nervous system,
but we did not observe changes in gene expression in rest mutant
brain. However, many Rest targets are robustly expressed in the
brain and are involved in a host of essential neural processes.
Discrete alteration of expression of these genes might have
profound consequences for higher-order neural functions. In
addition, recent studies of a conditional Rest knockout in the mouse
revealed a requirement for Rest in adult neurogenesis (Gao et al.,
2011). Regulation of gene expression in the adult brain may be a
primary Rest function. Future studies will explore the behavioral
consequences of Rest depletion.
Regulation of neural migration may be a principal function of
Rest. Abnormalities in FBMN migration were observed in our rest
mutant. In addition, the OPC deficiencies and secondary motor
neuron defects (in the NRCKgmc641 reporter line) might also stem
from altered migration. Adhesion molecules are abundant among
Rest targets (Otto et al., 2007), and Rest has been implicated in the
regulation of radial migration of mouse neocortical progenitors
(Mandel et al., 2011). Together, these data suggest a key role for
Rest in neuronal migration.
In conclusion, the zebrafish rest mutant is a valuable tool that
will help to broaden our understanding of Rest function. Our
analysis reveals that although Rest represses the expression of
RE1-containing target genes in several contexts, the effects often
appear to modulate the timing or levels of expression during
development rather than to silence vigorous expression. Although
we identified discrete requirements for Rest during development,
we found no evidence for a role as a fundamental regulator of
neurogenesis or cell fate during development. Examination of the
mutant also demonstrated that Rest-mediated repression of target
genes persists in non-neural tissues in adult animals. Additional
studies of the mutant will clarify the functions of Rest within the
nervous system and in non-neural tissue to establish a more
comprehensive view of Rest regulation of an expansive set of target
genes.
Acknowledgements
We thank the many members of the community who provided reagents;
Carolyn Milano and Azeez Aranmolate for experimental support; Ian Wood
and Sean McCorkle for assistance identifying RE1 sites; Bernadette Holdener,
Nurit Ballas and Joel Levine for comments on the manuscript; and Amanda
Levine, Won Seok Kwag and Erika Wunderlich for fish care.
Funding
This work was supported by NYSTEM [C026414 to H.I.S.]; National Institutes
of Health [HD066000 to H.I.S., DK064973 to V.E.P., R21 MH083614 to J.S.M.];
and March of Dimes Basil O’Connor [5-FY10-7 to J.S.M.]. Deposited in PMC
for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
DEVELOPMENT
3846 RESEARCH ARTICLE
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.080994/-/DC1
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