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First published online May 28, 2004
doi: 10.1242/10.1242/dev.01166
Centre for Developmental Genetics, School of Medicine and Biomedical Science, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK
E-mail: v.t.cunliffe{at}shef.ac.uk
Accepted 12 March 2004
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: Histone deacetylase, Chromatin, Neurogenesis, Zebrafish
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Artavanis-Tsakonas et al.,
1999). In vertebrate and invertebrate embryos, neuronal precursor
cells are born within small proneural clusters as a result of Notch- and
Delta-dependent lateral inhibitory interactions (reviewed by
Chan and Jan, 1999).
Delta-bound Notch protein is cleaved intracellularly and the intracellular
fragment promotes transcription of genes encoding transcriptional repressors
of the hairy/Enhancer of split [E(spl)] family. Within a Notch-expressing
cell, E(spl) proteins repress genes required for the adoption of a neuronal
fate, including components of the achaete-scute complex and other
bHLH genes such as neurogenin and neuroD. Homologues of
Drosophila neurogenic genes such as notch, delta and
hairy/E(spl) have been identified in many model vertebrates. In the
zebrafish, three delta homologues have been described
(Dornseifer et al., 1997;
Appel and Eisen, 1998;
Haddon et al., 1998), and
seven different Hairy/E(spl) (her) genes have been
characterised to date
(www.ensembl.org/Danio_rerio).
The adoption of a neuronal fate in vertebrate embryos involves the
sequential activation of bHLH proneural genes (reviewed by
Brunet and Ghysen, 1999;
Chitnis, 1999). In the mouse,
achaete-scute orthologues such as Mash1 (Ascl1 - Mouse
Genome Informatics) are required early on in neural cells to enable them to
acquire a neuronal precursor identity (Cau
et al., 1997). Expression of the murine bHLH genes neurogenin
(Ngn1) and Neurod1 (previously known as neuroD) requires
Mash1 activity (Cau et al.,
1997). In the zebrafish, both ash1a and ngn1 are
required for specification of epiphysial neurones
(Cau and Wilson, 2002).
ngn1 is also required for the formation of Rohon-Beard sensory
neurones in the spinal cord (Cornell and
Eisen, 2002), and later on in development for specification of
dorsal root sensory ganglia (Andermann et
al., 2002).
In the mouse embryo, the E(spl) protein Hes1 represses multiple
proneural genes. Targeted inactivation of Hes1 causes upregulation of
Mash1 and Ngn1, leading to accelerated neurogenesis and a
decrease in the number of later born neurones
(Ishibashi et al., 1995).
Conversely, ectopic expression of Hes1 prevents neuronal
differentiation (Ishibashi et al.,
1994). The zebrafish orthologue of Hes1, her6, is
expressed in the developing CNS (Pasini et
al., 2001), suggesting that it may perform a function related to
that of Hes1. Murine Hes5, in contrast to Hes1, is not required for
repression of Mash1 during early neurogenesis but instead functions
synergistically with Hes1 at a later stage to repress Ngn1
(Cau et al., 2000). The
zebrafish orthologue of Hes5, her4, similarly represses ngn1
in the neural plate (Takke et al.,
1999). Taken together, these studies indicate that different
E(spl) homologues perform distinct roles during vertebrate
neurogenesis. Increased understanding of the distinct functions of
E(spl) genes, together with a better appreciation of how they are
regulated, promise to yield important new insights into the molecular
mechanisms controlling neurogenesis.
Covalent chromatin modifications play key roles in regulating eukaryotic
gene expression (reviewed by Strahl and
Allis, 2000). The acetylation of core histones on N-terminal
lysines is a major determinant of the transcriptionally active state of many
genes and chromatin-associated histone acetyltransferases (HATs) are known to
perform essential functions in embryonic development. By contrast, the
recruitment of histone deacetylase (HDAC) enzymes to specific genes presages
their transcriptional silencing. The results of these and other studies
suggest that chromatin modifying enzymes may be involved in the establishment
and maintenance of cell memory during embryonic development (reviewed by
Turner, 2002). Indeed,
targeted deletion of murine hdac1 reduces embryonic growth, leading
to morphological abnormalities in the head and allantois
(Lagger et al., 2002).
However, few other insights into the roles of vertebrate HDACs in embryonic
development have emerged to date. Here, I have exploited a mutation in
zebrafish hdac1 to investigate the function of this gene in the
developing nervous system. The results reveal for the first time a primary, in
vivo requirement for hdac1 to maintain vertebrate neurogenesis and
evidence is presented that this occurs via repression of Notch targets,
including her6, the zebrafish orthologue of murine Hes1.
Moreover, I demonstrate that expression of proneural genes and neuronal
specification are severely impaired in distinct CNS territories of mutant
embryos within which strong ectopic expression of her6 is observed.
Although the hdac1 mutant hindbrain is segmented, the patterning of
post-mitotic neurones and glia within each rhombomere is disorganized. In
addition, hdac1 mutants fail to maintain the responsiveness of
hindbrain neural precursor cells to hedgehog signalling, which results in the
specification of very few branchiomotor neurones. Taken together, these
results reveal a surprisingly specific requirement for hdac1 to
maintain neurogenesis and enable neuronal fates to be realised in the
zebrafish CNS.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Homozygous hdac1hi1618 mutants were distinguished from siblings at 22-24 hours post-fertilization (hpf) by their reduced anterior hindbrain development. Homozygous smohi1640 mutants were distinguished from siblings at 24 hpf by their U-shaped somites. Homozygous mib mutants were distinguished from siblings by their abnormal trunk morphology.
hdac1 cDNA cloning
Full-length hdac1 cDNA clones were amplified by RT-PCR using the
forward primer 5'-ggc agg cgc agg ctg taa tt-3' and the reverse
primer 5'-atg cat cca gga gga ctg gc-3', based on the
hdac1 cDNA sequence deposited in the EMBL database (Accession Number,
AF506201).
Microinjection of morpholino antisense oligonucleotides and synthetic mRNA
A morpholino (MO; Gene Tools, LLC) was designed to block translation
initiation of hdac1 mRNA, using DNA sequence obtained from the EMBL
database (Accession Number, AF506201) and verified by independent cDNA
cloning: hdac1-MO: 5'-ttg ttc ctt gag aac tca gcg cca
t-3'. A second MO identical in sequence to hdac1-MO apart from
four mismatching nucleotides (highlighted in bold), was used to control for
non-specific effects of MO injection: Control-MO: 5'-ttg ctc
gtt gag aac tct gca cca t-3'
MOs were microinjected into zebrafish embryos at the one- to two-cell stage
in a volume of 
2 nl, at a final concentration of 0.3 mM in water. Neither
the control MO nor an irrelevant sequence MO known from previous studies to be
biologically inert (5'-cct ctt acc tca gtt aca att tat a-3')
exhibited any observable effects on embryonic development after
microinjection.
To modulate the expression level of shh in vivo, in vitro-synthesised capped mRNA encoding zebrafish Shh was microinjected into embryos at the one- to two-cell stage, at a dose of 100 pg/embryo along with the appropriate dose of MO.
Histology and immunohistochemistry
For histological analysis, embryos were fixed in 4% paraformaldehyde,
embedded in paraffin wax, then 8 µm sections were taken and stained with
Haematoxylin and Eosin. Immunohistochemistry was performed using standard
procedures (Schulte-Merker,
2003), which incorporated a 5 minute permeabilisation step with
ice-cold trypsin for embryos older than 28 hpf (omitted for Isl1 staining).
Antibodies were used at the following dilutions: anti-Isl1 monoclonal (39.4D5;
Developmental Studies Hybridoma Bank, Iowa, USA), 1:500; anti-Phospho-H3
polyclonal (Upstate), 1:500; anti-Hu (BD Biosciences), 1:500; anti-GFAP (a
kind gift of Dr J. Clarke, UCL) (Nona et
al., 1989), 1:160. Primary antibody binding was visualized with
peroxidase- or FITC-conjugated secondary antibodies.
RNA in situ hybridisation
Digoxigenin-labelled probes were prepared as recommended by the
manufacturer (Roche). Whole-mount in situ hybridisation was performed using
standard procedures (Oxtoby and Jowett,
1993). Details of the probes used are available on request.
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). However, few details of the defects caused by loss of
hdac1 function were described in that report, and so a systematic
analysis of the hdac1 mutant phenotype was initiated in order to gain
insights into the requirements for hdac1 function during embryonic
development. The expression pattern of hdac1 during embryonic
development was first documented using probes prepared from a full-length
hdac1 cDNA clone. Fig.
1 shows that hdac1 is maternally expressed, with
transcripts being detectable at all developmental stages analysed and in all
regions of the embryo. Particularly strong zygotic hdac1 expression
is evident in the anterior CNS of embryos at the 18-somite stage and 24 hpf,
suggesting that there might be a specific requirement for hdac1
function in the developing brain. As development continues, hdac1
transcripts selectively accumulate throughout the brain and spinal cord
(Fig. 1).
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) over a
period from 25 to 38 hpf (Fig.
3; Table 1). At 25
hpf, cell proliferation in the hdac1hi1618 mutant
hindbrain is significantly lower than that in unaffected siblings. However, by
33 hpf, the mitotic indices of hdac1hi1618 mutants and
siblings are similar and they remain so at 38 hpf
(Fig. 3; Table 1). Thus, although there
is a transient proliferation deficit in the hdac1hi1618
mutant hindbrain at 25 hpf, the normal proliferation rate of neural precursor
cells is subsequently regained and maintained independently of hdac1
function.
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) and ngn1 (Blader
et al., 1997) were compared in hdac1hi1618
mutants and siblings. At 25 hpf, expression of ash1b (ashb -
Zebrafish Information Network) and ngn1 is strikingly reduced
throughout the CNS of hdac1hi1618 mutants
(Fig. 4E-H), demonstrating that
neurogenesis is significantly compromised by loss of hdac1
function.
A Notch-activated repressor of proneural gene expression, her6, is derepressed in hdac1 mutants
In view of the evidence that hdac1 functions primarily in the
transcriptional repression of target genes (reviewed by
Ng and Bird, 2000), it seemed
reasonable to postulate that the downregulation of proneural gene expression
observed in hdac1hi1618 mutants could be an indirect
consequence of derepressing other genes that are themselves direct targets of
hdac1-mediated repression. Proneural genes are well-characterised targets of
Notch-dependent transcriptional repression by members of the hairy/E(spl)
family of bHLH proteins (reviewed by
Campos-Ortega, 1993;
Chitnis, 1999). In the
mammalian CNS, the Notch target gene Hes1 is required during
neurogenesis to co-ordinately repress transcription of both Mash1 and
Ngn1 (Cau et al.,
2000). This raised the possibility that its zebrafish orthologue,
her6 (Pasini et al.,
2001), might be aberrantly expressed in
hdac1hi1618 mutant embryos. At 26 hpf, her6 is
weakly expressed in the lateral hindbrain of wild-type embryos, but by 33 hpf,
transcripts have accumulated in two distinct lateral stripes running caudally
from the rhombic lip (Fig.
5A,C). In 26 hpf hdac1hi1618 mutant embryos,
her6 is expressed in the medial hindbrain, being particularly
abundant in rhombomeres 5 and 6, and this aberrant pattern persists at 33 hpf
(Fig. 5B,D). In wild-type
embryos, the proneural genes ash1b and ngn1 exhibit
distinct, partially overlapping expression patterns in the hindbrain
(Fig. 5G,I,M,O). However, in
the hdac1 mutant hindbrain, the abundance of ash1b and
ngn1 transcripts is dramatically reduced both at 26 hpf and 33 hpf
(Fig. 5H,J,N,P). Transverse
sections through rhombomere 5 further reveal the complementary nature of the
her6 expression domain compared with those of ash1b and
ngn1, in both hdac1hi1618 mutant and unaffected
sibling embryos (Fig. 5). Thus,
loss of hdac1 function causes an increase in her6 transcript
levels and suppresses expression of ash1b and ngn1
(Fig. 5E,F). In the dorsal
diencephalon of wild-type embryos, the expression domain of her6 is
also complementary to those of ash1b and ngn1
(Fig. 6), both at 26 hpf and at
33 hpf Moreover, the her6 expression domain is expanded in the dorsal
diencephalon of hdac1hi1618 mutants, whereas those of
ash1b and ngn1 are correspondingly reduced or eliminated, at
26 and 33 hpf (Fig. 6). Taken
together, these results demonstrate that hdac1 is required both to
repress her6 and to promote expression of ash1b and
ngn1 during the development of the hindbrain and dorsal
diencephalon.
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)
(Fig. 7). Strikingly, in the
hdac1hi1618 mutant hindbrain, Hu-expressing cells fail to
adopt the characteristic segmental arrangement in each rhombomere, and instead
they assemble into an unsegmented column on each side of the hindbrain
(Fig. 7A-F). Between 25 and 38
hpf, the number of Hu-positive cells within the
hdac1hi1618 mutant hindbrain progressively increases,
although in comparison to the hindbrain of sibling embryos, there are
consistently fewer neurones. As the hdac1hi1618 mutant
hindbrain develops, most of the newly born Hu-positive neurones accumulate in
rhombomeres 2, 3 and 4, whereas relatively few are found in rhombomeres 5 and
6 (Fig. 7E,F), in stark
contrast to the situation in wild-type embryos
(Fig. 7E,F). Glia are also
specified in the hdac1 mutant hindbrain but they, like the
Hu-expressing neurones, also fail to become organised into segmental arrays,
and are distributed throughout the hindbrain in predominantly lateral
locations, apparently intermingled with Hu-expressing neurones
(Fig. 7G,H). Taken together,
these results demonstrate that in the hindbrain hdac1 is specifically
required for the production and segmental patterning of both neurones and
glia.
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).
Epiphysial neurones express the Isl1 transcription factor once they have been
specified within the epithalamic region of the dorsal diencephalon
(Masai et al., 1997).
Branchiomotor and spinal motoneurones are induced by hedgehog signalling in
the hindbrain and spinal cord, respectively, and they also express Isl1
protein in response to such signals
(Chandrasekhar et al., 1997).
Intriguingly, homozygosity for the hdac1hi1618 mutation
eliminates Isl1 expression in the anterior half of the epiphysis
(Fig. 8A,B), and prevents
specification of almost all branchiomotor neuronal precursors except for two
small clusters of trigeminal (nV) neurones in rhombomere 2 and two small
groups of facial (nVII) precursors located in rhombomere 4
(Fig. 8C,D). To determine
whether the Isl1-positive cells that were specified in the hindbrain of
hdac1-deficient embryos were able to differentiate fully into
motoneurones with axons, a morpholino targeted against the translation start
site of hdac1 mRNA was microinjected into embryos specifically
expressing an Isl1-green fluorescent protein (GFP) transgene in branchiomotor
neurones (Higashijima et al.,
2000). At 30 hpf, hdac1-MO-injected embryos exhibited a
pattern of four small clusters of Isl1-positive branchiomotor neurones
identical to that observed in hdac1hi1618 mutants, two of
which (nV neurones) were located in rhombomere 2, and two of which (nVII
neurones) were located in rhombomere 4 (compare
Fig. 8C,D with 8E-H; and see
Table 2). Moreover, whereas the
population of GFP-positive motoneurones in control-MO injected embryos was
considerably increased in size between 30 hpf and 36 hpf
(Fig. 8E,F), the rudimentary
pattern of four small clusters of GFP-positive branchiomotor neurones remained
unchanged in hdac1-MO-injected embryos
(Fig. 8G,H). No GFP-positive
cells were ever observed in the caudal part of the hindbrain that normally
gives rise to nIX and nX motoneurones. Nevertheless, the GFP-positive
motoneurones that were present in hdac1-MO-injected embryos did
appear to differentiate normally and axon outgrowth could be clearly observed
(Fig. 8G,H). These results
demonstrate that relatively few branchiomotor neurones are specified in
hdac1-deficient embryos, which remain spatially restricted to
rhombomeres located anterior to rhombomere 5, and they do not undergo
tangential migration posteriorly. Remarkably, however, these cells
differentiate into motoneurones with axons that project anteriorly.
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her6 is derepressed, proneural gene expression is extinguished, and neuronal specification is impaired in hdac1-deficient embryos, independently of Notch signalling
Notch signalling is essential for proper transcriptional activation of many
E(spl) genes during neurogenesis (for a review, see
Chitnis, 1999). In vitro
studies indicate that Notch-mediated transcriptional activation of
E(spl) genes is antagonised by a protein complex that contains Hdac1
(Kao et al., 1998). However,
the in vivo requirements for hdac1 functions in repression of
E(spl) genes, particularly in relation to the effects of Notch
signalling, have not been defined. The mind bomb (mib)
mutation profoundly impairs Notch signalling such that extensive, premature
neuronal differentiation occurs throughout the embryonic CNS
(Itoh et al., 2003). To test
the hypothesis that hdac1 function is required for repression of
her6 in the developing CNS, and that Notch signalling is required to
relieve this repression, her6 expression was analysed in the
mib mutant under conditions where levels of hdac1 activity were
either unperturbed or reduced by hdac1-MO microinjection.
Homozygosity for the mib mutation significantly reduces the abundance
of her6 transcripts both in the dorsal diencephalon and the hindbrain
(Fig. 9), confirming that notch
signalling is essential for proper expression of her6. In stark
contrast, microinjection of the hdac1-MO into either mib
mutant or sibling embryos caused a dramatic derepression of her6 both
in the dorsal diencephalon and in hindbrain rhombomeres 5 and 6
(Fig. 9, arrows;
Table 3). These results
demonstrate that hdac1 does indeed act as a repressor of her6 in the
hindbrain and dorsal diencephalon, and also that the repressive effect of
hdac1 on her6 is normally alleviated by Notch signalling. In further
confirmation of these findings, expression of the proneural gene ngn1
in the CNS was strictly dependent on wild-type levels of hdac1
activity, irrespective of whether hdac1-deficient embryos were
homozygous for the mib mutation or not
(Fig. 9I,L). Finally,
immunostaining for Isl1 protein revealed that loss of hdac1 function
severely impaired neuronal specification in both the epiphysis and the
hindbrain in a mib-independent manner
(Fig. 9M-T;
Table 3). Reduced levels of
hdac1 eliminated Isl1 expression in the anterior epiphysis, both in
mib siblings and mutants (Fig.
9M-P). The hindbrain of hdac1 morphants developed with
two pairs of Isl1-positive cell clusters in r2 and r4 and none posterior to
r4, as was observed in hdac1hi1618 mutants
(Fig. 9S). By contrast,
supernumerary Isl1-positive cells were found throughout the hindbrain of
mib mutants (Fig. 9R).
However, microinjection of the hdac1-MO into mib mutant
embryos suppressed this widespread, ectopic neurogenesis and instead
restricted the specification of Isl1-positive cells to the two pairs of cell
clusters in rhombomeres 2 and 4 that are characteristic of
hdac1hi1618 mutants
(Fig. 9T).
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; Varga et al.,
2001; Lewis and Eisen,
2001). It was therefore of interest to investigate the
relationship between hedgehog signalling and the requirement for
hdac1 function in motoneurone specification. In situ hybridisation
analysis for expression of shh and the shh target gene ptc1
revealed that hdac1 does not play a major role in determining the
expression patterns of these genes in motoneurone-forming regions because
shh is expressed in ventral midline cells in both
hdac1hi1618 mutant and sibling embryos, and expression of
the shh target ptc1 is relatively unperturbed both in the CNS and
paraxial mesoderm (Fig.
10A-D). A localised reduction in expression of both shh
and ptc1 is found more anteriorly, however, in the zona limitans
intrathalamica of the forebrain (zli, Fig.
10A-D). In wild-type embryos, the level of shh expression
is strictly limiting for the number of branchiomotor neurones that are
specified because over-expression of shh induces supernumerary
branchiomotor neurones (Chandrasekhar et
al., 1998). Although specification of the majority of
branchiomotor neurones requires hdac1, small clusters of
hdac1-independent motoneurones do form in rhombomeres 2 and 4 of
hdac1-deficient embryos. Therefore, to determine whether shh
overexpression could expand the population of branchiomotor neurones formed in
hdac1-deficient embryos, shh mRNA was microinjected into
Isl1-GFP transgenic embryos with either a hdac1-MO or a control MO.
Microinjection of shh mRNA along with the control MO induced many
supernumerary branchiomotor neurones in the hindbrain of Isl1-GFP embryos.
However, no supernumerary branchiomotor neurones were formed when the
hdac1-MO was co-injected with shh mRNA and instead only the
rudimentary pattern of branchiomotor neurones that is characteristic of
hdac1hi1618 mutants was observed
(Fig. 10;
Table 4). Thus, although the
hedgehog pathway is active in hdac1-deficient embryos
(Fig. 10A-D) and there are
many mitotically active cells in the hindbrain
(Fig. 3; Table 1), very few
Isl1-expressing motoneurones are produced in response to hedgehog signals
(Fig. 10E-L). To confirm that
the branchiomotor neurones that did form in hdac1-deficient embryos
were nevertheless dependent on hedgehog signalling, the effect of knocking
down hdac1 function in smoothened mutants
(Chen et al., 2001;
Varga et al., 2001) was
analysed. At 26 hpf, the first cells to detectably express Isl1 protein in the
hindbrain of control-MO-injected, unaffected sibling embryos were a small
group of rostrally located trigeminal (nV) motoneurones in rhombomere 2
(Fig. 11A). Although loss of
smoothened function completely abolishes the specification of these
first-detected branchiomotor neurones
(Fig. 11C), loss of
hdac1 function had no observable effect on their formation
(Fig. 11B). By 32 hpf,
control-MO-injected, unaffected siblings had developed a properly expanded and
appropriately positioned set of branchiomotor neurones
(Fig. 11E), whereas once more,
no Isl1-positive cells were detectable in the hindbrain of smoothened
mutants (Fig. 11G). As
observed previously, at 32 hpf hdac1-deficient embryos developed only
the two further small clusters of facial (nVII) motoneurones in r4 in addition
to the trigeminal (nV) population first detected in r2 at 24 hpf
(Fig. 11F), all of which were
strictly smoothened-dependent
(Fig. 11H). Taking the results
described in Figs 10 and
11 together, it can be
concluded that the hedgehog signalling pathway is intact in hdac1
mutants, but sustained production of hedgehog-dependent motoneurones, although
initially normal, is not efficiently maintained even when the level of
hedgehog expression is experimentally increased. Therefore, consistent with
its function as an antagonist of Notch signalling, these results reveal that
hdac1 is required in the hindbrain to maintain the production of
branchiomotor neurones in response to hedgehog signalling.
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Discussion |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). In Drosophila, a loss-of-function mutation in the
hdac1 orthologue Rpd3 causes a pair-rule phenotype
(Mannervik and Levine, 1999),
whereas in C. elegans, mutation of hda-1 causes
post-embryonic defects in gonadogenesis and vulval development
(Dufourcq et al., 2002). In
zebrafish, mutation of hdac1
(Golling et al., 2002) causes
a phenotype in which, as shown here, distinct aspects of the development of
several organs and tissues are affected.
Hdac1 as a repressor of Notch targets in neural precursor cells
Histological analysis of the CNS in hdac1 mutants initially
indicated that neurogenesis may be sensitive to loss of hdac1
function. The ensuing analysis demonstrated unequivocally that hdac1
is indeed required to promote neurogenesis and evidence is presented that this
is accomplished by transcriptional repression of Notch-activated target genes
such as her6. Her6 is the orthologue of mammalian Hes1,
which is required for Notch-driven repression of Mash1 and
Ngn1 (Cau et al.,
2000). Consistent with these observations, transcripts of the
proneural genes ash1b and ngn1 were almost completely absent
in hdac1-mutant embryos, suggesting that, as for Hes1 in mammals,
Her6 is a specific repressor of multiple proneural genes. Derepression of
her6 was greatest in the dorsal diencephalon and hindbrain
rhombomeres 5 and 6, two regions of the CNS where neuronal specification, as
revealed by Isl1 immunostaining, was particularly severely affected
(Fig. 8). Intriguingly,
her6 derepression caused by hdac1 deficiency was epistatic
to the mind bomb neurogenic phenotype, both in the dorsal
diencephalon and in rhombomeres 5 and 6 of the hindbrain. These observations
unequivocally show that a function of Notch signalling is to relieve
hdac1-mediated transcriptional repression of Notch targets in the
CNS, which is consistent with studies in other species
(Kao et al., 1998;
Barolo and Posakony, 2002).
Interestingly, loss of hdac1 function did not cause widespread
derepression of her6 throughout the embryo. Instead, her6
derepression was strongest in CNS territories undergoing neuronal
specification, implying that hdac1-mediated transcription silencing is
selective for neural patterning processes, perhaps through interactions with
neural-specific repressors. The results described here also imply that the
products of Notch target genes such as the her6 transcriptional repressor can
function in an hdac1-independent manner, possibly through redundant
interactions with other class I HDACs.
In Drosophila, Notch target genes are actively repressed by
DNA-bound Suppressor of Hairless [Su(H)], which recruits co-repressor
molecules such as groucho and CtBP to the target locus via interactions with
the Hairless protein (Barolo et al.,
2002). Other studies in cultured mammalian cells previously found
that the Su(H) orthologue CBF1 bound the HDAC1-containing SMRT complex
(Kao et al., 1998). Moreover,
in Xenopus animal cap explants, the broad specificity HDAC inhibitor
trichostatin A caused a two-fold increase in expression of the Notch target
gene ESR1 (an orthologue of zebrafish her4) in response to
overexpression of Xdelta1 mRNA
(Kao et al., 1998), suggesting
a role for histone deacetylase in repression of Notch targets. The experiments
reported here extend these observations significantly by demonstrating that in
the zebrafish embryonic CNS, hdac1 is required for repression of
Notch targets in a manner that still renders these genes inducible by Notch
signalling. No vertebrate orthologue of the hairless gene has yet
been described, and the SMRT protein can interact directly with the orthologue
of Su(H) (Kao et al., 1998).
However, proteins such as SHARP and SKIP may also modulate connectivity
between DNA-bound Su(H) and Hdac1 in vertebrate embryos
(Oswald et al., 2002). It will
now be of great interest to determine whether interactions between Hdac1,
SHARP, SKIP and Su(H) orthologues are essential for repressing Notch targets
during neurogenesis in the zebrafish embryo.
A widespread requirement for hdac1 in neuronal specification and patterning
Immunostaining for expression of Hu proteins and GFAP revealed that
formation of neurones and glia was highly abnormal in the hindbrain of
hdac1 mutant embryos (Fig.
7). There are fewer post-mitotic neurones in hdac1 mutant
embryos than in siblings at each of the three stages analysed, and the
characteristic segmental organisation of these neurones with their associated
glia is lost. However, loss of hdac1 function does not cause a
general arrest of CNS growth and development, because although cell
proliferation in the hdac1 mutant hindbrain is reduced at 25 hpf in
comparison to the situation in sibling embryos, it regains its normal
proliferative capacity by 33 hpf and a similarly high level of mitotic
activity persists at 38 hpf. Moreover, throughout the period from 25 hpf to 38
hpf, the size of the Hu-positive neuronal population in the hindbrain
progressively increases. It is possible that the transient reduction in cell
proliferation within the hdac1 mutant hindbrain specifically affects
a particular step of neurogenesis, because the defects in neurogenesis are
irreversible and become more profound with time, whereas cell proliferation
within the hindbrain recovers. It is also possible that hdac1 mutants
selectively accumulate proliferating neural precursors as a direct consequence
of derepressing Notch target genes
(Solecki et al., 2001). Future
experiments will investigate these possibilities. However, taken together with
the finding that patterning markers such as epha4 are properly
segmentally expressed in the hindbrain
(Fig. 4), the observed
abnormalities clearly illustrate that hdac1 is required to
efficiently couple neurogenesis to the mechanisms determining segmental
patterning of the hindbrain.
Hdac1-deficient embryos exhibited a range of defects in
specification of neuronal subtypes, as revealed by immunostaining for Isl1 and
by confocal microscopy of an Isl1-GFP transgenic line. In the dorsal
diencephalon of hdac1 mutants, there was a striking deficit of
anterior epiphysial neurones in a position corresponding to the diencephalic
territory within which strong expression of her6 and extinction of
proneural gene expression was observed (Figs
6,
8). In the hindbrain, nascent
clusters of Isl1-positive trigeminal (nV) and facial (nVII) motoneurones were
produced in rhombomeres 2 and 4 of hdac1-deficient embryos, but these
clusters failed to expand properly and no additional branchiomotor neurones
were formed. Nevertheless, those nV (r2) and nVII (r4) motoneurones that were
specified persisted in their original positions within the hindbrain and they
produced correctly oriented axons that are characteristic of properly
differentiated neurones. Interestingly, there was no evidence of tangential
migration caudally (Chandrasekhar et al.,
1997), by branchiomotor neurones born in rhombomere 4, into
rhombomeres 5 and 6 of the hdac1 mutant hindbrain, where
her6 was strongly expressed. It remains unclear whether this
abnormality solely reflects a tangential migration defect in nVII neuronal
precursors of hdac1 mutants that were born in rhombomere 4, or
whether the observed defect is also the consequence of a failure to specify
nVII motoneurones from separate precursors originating in rhombomeres 5 and
6.
Within the trunk, loss of hdac1 function caused a substantial reduction in the size of the spinal motoneurone population throughout the spinal cord, as revealed by Isl1 immunostaining, and a milder effect on the number of Rohon-Beard cells was also evident (Fig. 8), indicating that neuronal specification can be initiated throughout the length of the spinal cord but in the absence of hdac1 function it is not efficiently maintained.
hdac1 facilitates the response of neuronal precursors to hedgehog signalling and the acquisition of motoneurone identity
All branchiomotor neurones require hedgehog signalling for specification
and at the onset of this developmental process, hdac1-deficient and
wild-type embryos were indistinguishable
(Fig. 11). However, later
phases of branchiomotor specification were defective in
hdac1-deficient embryos, and this could not be averted by
overexpression of shh (Figs
10,
11). Thus, hdac1 is
required to maintain the response of neural precursors in the hindbrain to
hedgehog signals. Impairment of this response could be a direct consequence of
increased Notch target gene expression in hdac1-deficient embryos.
Previous work demonstrated that expression of the proneural gene ngn1
in the developing CNS is positively regulated by hedgehog signalling
(Blader et al., 1997), raising
the possibility that loss of hdac1 function directly inhibits the
response to hedgehog signalling by preventing proneural gene expression.
Recent studies in Drosophila have demonstrated that Notch signalling
also prevents the hedgehog-mediated activation of collier in the wing
margin (Glise et al., 2002).
As vertebrate homologues of collier have previously been implicated
in control of neurogenesis (Bally-Cuif et
al., 1998; Dubois et al.,
1998), it is conceivable that derepression of Notch targets such
as her6 in the hdac1 mutant hindbrain could directly inhibit
hedgehog-mediated expression of collier homologues. This possibility
will now be investigated. Hdac1 protein has been found in physical association
with numerous transcriptional repressors, and may be required for the
deacetylation of histones associated with many different target genes in
neural cells. Nevertheless, the results presented here unveil her6 as
a likely direct target of hdac1-mediated transcriptional repression
and imply that the her6 locus is hyperacetylated in hdac1
mutant embryos. Deacetylated core histones are substrates for
lysine-methylation by a large family of SET-domain-containing histone
methyltransferases (for a review, see
Turner, 2002), and so the
her6 locus of wild-type embryos may also exhibit histone methylation
patterns that are under-represented in hdac1 mutant embryos. These
possibilities will now be investigated.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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