1 Institut für Entwicklungsbiologie, Universität zu Köln, 50923
Köln, Germany
2 IGBMC, Parc d'Innovation, BP163, 67404 Illkirch Cedex, France
¶ Authors for correspondence (e-mail: stefan{at}uoneuro.uoregon.edu and Eberhard.Rudloff{at}uni-koeln.de)
Accepted 12 March 2004
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SUMMARY |
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Key words: Zebrafish, neurog1 (ngn1), Her3
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Introduction |
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The Notch signalling pathway is similarly organised in vertebrates (for a
review, see Lewis, 1996). As
in Drosophila, one of the functions of the pathway is to select
individual cells for specific fates from groups of initially equivalent cells
(Chitnis et al., 1995
;
Henrique et al., 1995
;
Chitnis and Kintner, 1996
;
Dornseifer et al., 1997
;
Wettstein et al., 1997
;
Appel and Eisen, 1998
;
Appel et al., 2001
;
Haddon et al., 1998
;
Takke et al., 1999
).
Injections of mRNA encoding variants of components of the Notch pathway have
provided evidence for a regulatory feedback loop, organised similar way to
that described for Drosophila, in both Xenopus and zebrafish
(Wettstein et al., 1997
;
Takke et al., 1999
).
Activation of Notch receptors leads to activation of E(spl)
homologues (Jarriault et al.,
1995
; Tamura et al.,
1995
; Hsieh et al.,
1996
; Kopan et al.,
1996
; Wettstein et al.,
1997
; Schroeter et al.,
1998
; Takke and Campos-Ortega,
1999
; Takke et al.,
1999
) mediated by CSL proteins
(Wettstein et al., 1997
).
Thus, in mice, a loss-of-function mutation in the RBP-J
gene
(Rbpsuh Mouse Genome Informatics), the Su(H)
homologue, leads to repression of Hes5 and, as a consequence, to upregulation
of the proneural gene Math4a (Neurog2 Mouse Genome
Informatics) (de la Pompa et al.,
1997
). In zebrafish, this feedback loop operates on the proneural
gene neurogenin 1 (neurog1; previously known as
ngn1) (Blader et al.,
1997
), the product of which binds to E-boxes in the promoter of
the Delta homologue deltaD, and activates its transcription
(Hans and Campos-Ortega,
2002
).
We describe here a new zebrafish E(spl) homologue, her3, the expression of which is restricted to neural territories, where it represses transcription of the proneural gene neurog1. Gel retardation assays show that Her3 binds specifically to N-boxes in the promoter regions of neurog1 and of her3, thus contributing to its own regulation. The function of her3, like that of other members of the E(spl) family, depends on Notch signalling. However, her3 differs from the other family members in that its transcription is repressed rather than activated by Notch1a signalling.
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Materials and methods |
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Molecular cloning of her3 and UAS plasmid construction
PCR using degenerate primers was performed on reverse-transcribed total RNA
from zebrafish embryos at the 90%-epiboly to two-somite stage. PCR fragments
encoding peptides with similarity to Drosophila bHLH proteins were
used to screen a zebrafish cDNA library prepared in ZAP (Stratagene)
from RNA isolated from 3-15 hour zebrafish embryos (gift from C.
Fromental-Ramain and P. Chambon, Strasbourg). The GenBank Accession Number for
the her3 cDNA sequence is X97331. A genomic her3 clone (15
kb) was obtained from a genomic DNA library (`Easy-to-handle eukaryotic
genomic library' (zebrafish) Mo Bi Tec, Göttingen). The Accession Number
for the her3 genomic sequence is AY277702. The upstream sequence was
subcloned into pBsGAL4 (Scheer
and Campos-Ortega, 1999
). The plasmid pBs2xMARher4 was
generated by excision of the Notch1a:intra coding sequence from pBs2xMAR
notch1a-intra with SmaI
(Scheer and Campos-Ortega,
1999
). The gap was filled with the EcoRI digested and
blunt-ended coding sequence of the her4 cDNA
(Takke et al., 1999
). To
generate stable transgenic lines, plasmid DNA preparations and injections were
carried out following the procedures described by Scheer and Campos-Ortega
(Scheer and Campos-Ortega,
1999
).
pCS2+her3:gfp was made by PCR using the plasmid pCS2+her3 diluted 1:10 as template (see her3 for and her3 rev primers in Tables S1-S3 at http://dev.biologists.org/supplemental). The amplified 680 bp fragment was cut with EcoRI and BamHI, cloned in the pCS2+EGFP vector previously cut with the same enzymes and sequenced.
RNA injections
Eight constructs encoding Her3 variants and two Her3 fusion proteins were
generated by PCR using different 5' and 3' primers, and cloned
into the pCS2+ (Turner and
Weintraub, 1994). The coding sequence was amplified from the
her3 cDNA using her3 specific primers. The primers used for
constructs with terminal deletions (see Tables S1-S3) provided an artificial
ATG for the construct her3
N, an artificial WRPW motif
and stop codon for construct her3
C, and an artificial
stop codon for construct her3
WRPW. PCR products were
digested with the appropriate restriction enzymes and cloned into the
pCS2+ vector. In order to remove the endogenous stop codon to permit
the generation of the her3 fusion proteins, the coding sequence of
her3 was amplified with the primers SP6 and her3 fusion (including a
BamHI site), digested with EcoRI and BamHI, and
cloned into the EcoRI/BamHI sites of the vectors
pCS2+VP16 and pCS2+eng, which supply the sequences encoding
the VP16 transactivation and the engrailed repressor domains, respectively, in
the correct reading frame. The primer combinations used for the generation of
the constructs and fusion proteins are listed in Tables S1-S3 at
http://dev.biologists.org/supplemental.
Capped RNA was synthesised in vitro by transcription with SP6 polymerase from
the constructs described above, or from a pCS2-nuclear ß-Galactosidase
(nßgal) template DNA, using a Message Kit from Ambion.
The RNA was injected in a volume of 5 nl into one of the first two
blastomeres. In most cases, lacZ mRNA was co-injected at
concentrations previously shown to be innocuous for the zebrafish embryo
(Takke et al., 1999).
ß-Galactosidase was detected by antibody staining.
Morpholino injections
Morpholino-modified antisense oligonucleotides directed against
her3 (Gene Tools; MO her3
5'-TGCAGCCATTGTCCTTAAATGCTCA, 2 blocker
5'-TTAAAAAATCCAGATGAATAAGGAC-3') and the mismatch morpholino
5'-TGGAGGCATTGTGCTTAAATCCTGA-3' were injected at the one- to
four-cell stage at a concentration of 50-200 µM in 1xDanieau
(Nasevicius and Ekker, 2000).
Morpholinos were injected together with 0.2% Texas Red in a total volume of
5-10 nl. As an additional control, mRNA encoding a her3-gfp fusion
was co-injected with the morpholinos.
RT-PCR
Total RNA was extracted from 50 wild-type embryos, or 50 embryos injected
with MO her3, using the RNA-CleanTM System (Angewandte
Gentechnologie Systeme GmbH). Before precipitation, the RNA was treated with
2U of DNase (Boehringer Mannheim) for 30 minutes. Two µg of RNA was reverse
transcribed using Superscript-RT (Gibco-BRL) and 100 ng of random hexamers
(Boehringer Mannheim) in a 20 µl reaction, and 0.5 µl of this reaction
was subjected to PCR. As an internal control, we used primers that amplified a
400 bp fragment of the gene for elongation factor e-IF4a. After 3 minutes at
95°C, amplification was carried out for 1 minute at 95°C, 1 minute at
58°C and 1 minute at 72°C (27 cycles for her3 and 28 for
neurog1), with a final extension step for 10 minutes at 72°C. The
primers are given in Tables S1-S3 (see
http://dev.biologists.org/supplemental).
Gel retardation assays
Protein preparation followed the protocol described previously
(Chang et al., 1997). For each
experimental determination, three lanes were loaded with increasing
concentrations of protein (refer to the legends of Figs
3,
4 and
7). The specificity of binding
was tested in competition assays with unlabelled oligonucleotide. Binding
reactions were performed in the presence of oligonucleotides containing N
boxes, one of the DNA sequences recognised by E(spl) proteins (consensus
CACNAG) (Sasai et al., 1992
;
Tietze et al., 1992
;
Oellers et al., 1994
), from
the promoter region of her3 and neurog1
(Blader et al., 2003
).
Band-shift assays were carried out according to Fried and Crothers (Fried and
Crothers, 1984a; Fried and Crothers,
1984
) and Hendrickson and Schleif
(Hendrickson and Schleif,
1984
). The sequences of the oligonucleotides used in binding
assays were as follows (N-boxes are in bold, mutations are underlined).
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her3 promoter
In situ hybridisation and histological methods
Hybridisation of digoxigenin-labelled RNA probes to embryo whole mounts was
performed as described previously (Bierkamp
and Campos-Ortega, 1993). Embryos injected with RNA were prepared
for in situ hybridisation and for antibody staining, as described by
Dornseifer et al. (Dornseifer et al.,
1997
). For sectioning, embryos were embedded in Araldite
(Serva).
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Results |
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her3 is expressed within neural territories
Whole-mount in situ hybridisation revealed that her3 RNA is
expressed only in neural territories during embryogenesis
(Fig. 1). Expression was first
detected at about 30% epiboly in a coherent patch of cells within the dorsal
region of the epiblast, in a region corresponding to the prospective anlage of
the neural plate (Fig. 1A) (Woo
and Fraser, 1995). At 80% epiboly, transcripts disappear from the medial
region, splitting the primary domain into two
(Fig. 1B,C). By the tail-bud
stage, each of these has evolved into two extended longitudinal expression
domains, which progressively separate from each other during the formation of
the first somites. One of the domains is located rostrally and will eventually
split into several groups of her3-positive cells distributed
throughout the mesencephalon and rhombencephalon
(Fig. 1E-H). Double in situ
hybridisation using probes for egr2b and her3, pax2a and
her3, or her5 and her3
(Fig. 1I-L) show that the
anterior margin of the her3 domain corresponds to the anterior margin
of the mesencephalic primordium (Lun and
Brand, 1998) extending to rhombomere 5. At the tail-bud stage, the
her3 transcription domain ends cranially at the anterior border of
the her5 domain, which itself initially extends throughout the
midbrain/hindbrain anlage (Müller et
al., 1996
; Bally-Cuif et al.,
2000
), but is later restricted to the so-called intervening zone
at the midbrain/hindbrain boundary (Geling
et al., 2003
). Therefore, later in development, the cranial margin
of the her3 transcription domain continues to define the anterior
margin of the mesencephalon, whereas the her5 domain is located
further caudal. However, the her3 transcription pattern within the
mesencephalic/rhombencephalic region is very dynamic and no attempt was made
to define the different groups of her3-transcribing cells in this
region.
|
To characterise the regulatory region of the her3 gene, a 4.7 kb
segment of the 5' upstream DNA was fused to the GAL4 coding sequence,
and transformed into the germline of wild-type embryos. Three independent
chromosomal insertions were recovered, of which only one was able to
transactivate UAS:notch1a-intra. In situ hybridisation with a
gal4 cDNA probe showed that the gal4 transcription pattern
in the transgenic embryos reflects, with minor deviations, the expression of
the endogenous her3 gene (Fig.
2A-C). Thus, the expression of the gal4 transgene in
mesencephalic and rhombencephalic regions is slightly delayed relative to that
of her3, and the density of gal4 transcripts is higher than
in the case of endogenous her3 RNA. The transgene is also expressed
in the otic placodes, unlike the endogenous her3 gene. It is not
clear whether these differences are due to position effects. For our present
purposes, though, it is important to note that the 4.7-kb DNA that drives
transcription in the intermediary region of the neural plate at the level of
the spinal cord contains two high-affinity N boxes and a single high-affinity
Su(H) binding site (TGTGAGAA) (Bailey and
Posakony, 1995; Lecourtois and
Schweissguth, 1995
; Rebeiz et
al., 2002
). There are no low-affinity binding sites for
E(spl)-related proteins in this DNA fragment.
|
her3 is a repressor of the proneural gene neurog1
Embryos injected at the two-cell stage with her3 mRNA encoding the
full-length protein were collected at the one- to three-somite stage and
tested by in situ hybridisation for the expression of various other genes
(neurog1, deltaA, islet1 and elavl3). After injection of
her3 mRNA, a large proportion of embryos showed a pronounced
asymmetry in the neural plate, which was considerably broader within the
ß-galactosidase-expressing territory
(Fig. 3,
Table 1). The same effect has
been reported after misexpression of a Xenopus Notch variant
(Coffman et al., 1993), and
following misexpression of deltaD
(Dornseifer et al., 1997
) and
her4 (Takke et al.,
1999
) in the zebrafish. The significance of this effect on the
size of the neural primordium is unclear. In most embryos that show an
enlargement of the neural plate, there is a concomitant reduction in the
numbers of cells expressing markers of primary neurones (islet1 and
elavl3, Fig. 3A-C, not
shown). As her3 is not normally expressed in the regions of the
neural plate from which primary neurones originate, we asked whether this
effect is due directly to the product of the injected her3 mRNA, or
is caused by the products of other genes, the activity of which may depend on
Her3, and which are expressed in the territories in which primary neurones
arise. The proneural gene neurog1 elicits ectopic development of
islet1-positive cells (Blader et
al., 1997
; Takke et al.,
1999
) and is repressed by Her4, another member of the hairy-E(spl)
family (Takke et al., 1999
).
Thus, it is conceivable that the reduction in the number of primary neurones
seen in the injected embryos is due to repression of neurog1. Indeed,
probing with neurog1 of embryos injected with her3 mRNA
revealed that similar proportions of embryos have an enlarged neural plate and
a reduced density of neurog1 transcripts
(Fig. 3D,E). This observation
suggests that the effect of misexpression of her3 on primary neurones
is, at least in part, due to repression of neurog1. Injection of
her3 mRNA also weakly suppresses transcription of deltaA
(not shown), a target of neurog1
(Takke et al., 1999
). Gel
retardation assays suggest that repression of neurog1 transcription
may be mediated by direct binding of Her3 protein to N-boxes present in the
neurog1 promoter (CTC ACA AGC TCA CAC
GAG CTG) at position 129 relative to the ATG
(Fig. 3F)
(Blader et al., 2003
). Thus,
when increasing amounts of Her3 protein are incubated in the presence of
oligonucleotides containing N-boxes from the neurog1 promoter region
(see Materials and methods), clear shifts in the electrophoretic mobility of
the labelled probe are detectable. The shift can be selectively inhibited
either by adding non-radioactive wild-type oligonucleotides, or using
oligonucleotides containing mutations in both N-box sequences. In the latter
case, the affinity of the nucleotide for Her3 is considerably reduced, whereas
mutation of either box has a weaker effect. Furthermore, non-radioactive
mutant oligonucleotides do not effectively compete with the labelled wild-type
probe for binding of Her3.
|
Double in situ hybridisation with egr2b showed that
ectopic induction of neurog1 is restricted to rhombomeres 2 and 4
(Fig. 4B), encompassing the
region that connects the clusters of motoneurones and sensory neurones
(Geling et al., 2003). Ectopic
expression of neurog1 is associated with ectopic activation of a
number of genes involved in lateral inhibition (deltaA, deltaD, coe2
and her4) in the same regions of rhombomeres 2 and 4
(Fig. 4C-E, not shown), and
ectopic neurones can be subsequently detected within these same regions
(Fig. 4D). The available
evidence indicates that all these genes may be activated by Neurog1
(Bally-Cuif et al., 1998
;
Dubois et al., 1998
;
Takke et al., 1999
;
Hans and Campos-Ortega, 2002
)
and therefore, their ectopic activation in rhombomeres 2 and 4 might well be
caused by the ectopic induction of neurog1, leading to the formation
of ectopic neurones.
As reproducible phenotypic effects of blocking her3 mRNA translation with morpholinos were rather weak, i.e. restricted to a fairly small region of the neural anlage, whereas the her3 transcription domain is much broader, we suspected that the efficacy of the injected morpholinos might be compromised. To test whether the morpholinos can completely knock-down her3 activity, a construct encoding a Her3:gfp fusion was synthesised, and 400 ng/µl mRNA transcribed from this plasmid was injected together with each of the two morpholinos. Another batch of embryos was injected with 400ng/µl her3:gfp mRNA without the morpholinos. After injection, embryos showing Texas Red staining were selected, and the number of embryos with GFP-mediated fluorescence was determined between 30% and at 85% epiboly. 100% of the embryos in the control series showed a fluorescent signal (Fig. 5). The simultaneous injection of either morpholino completely eliminated the fluorescent signal. This result suggests that both morpholinos completely inhibit the translation of her3 RNA.
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Functional dissection of Her3
To gain insight into the structural requirements for Her3 function, we
constructed seven deletion variants of the her3 cDNA (see
Fig. 6), and injected the
corresponding mRNAs into embryos at the two-cell stage. Injected embryos were
processed for in situ hybridisation with a neurog1 probe. The effects
of mRNA injections were comparable for five of the seven variants,
her3N,
her3
b,
her3
bHLH,
her3
HLH and
her3
WRPW, which encode derivatives that
lack the LCC (low compositional complexity) domain, the basic domain, the bHLH
domain, the HLH domain and the C-terminal tetrapeptide WRPW, respectively. All
these variants have partially lost the ability to suppress neurog1
transcription (Table 1). By
contrast, the last two variants tested,
her3
orange and
her3
C, which encode products that lack the
so-called orange domain (Dawson et al.,
1995
) and a C-terminal segment, respectively, behave like
gain-of-function mutants. Injection of mRNA for either construct leads to a
large increase in the number of embryos that show reduced neurog1
transcription.
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|
Gel retardation assays suggest that Her4-mediated repression of
her3 occurs by direct binding of Her4 to N-boxes
(Tietze et al., 1992;
Sasai et al., 1992
;
Oellers et al., 1994
) present
in the her3 promoter (see Materials and methods). Thus, incubation of
increasing concentrations of Her4 protein in the presence of oligonucleotides
containing N-boxes from the her3 promoter region results in clear
shifts in the electrophoretic mobility of the labelled probes. The shifts
suggest binding of oligomeric forms of Her4 to the target DNA
(Fig. 8H).
We tested to what extent transcription of other zebrafish genes of the hairy/E(spl) family, such as her5 and her6, is also dependent on Notch signalling. To do this, we probed embryos from both of the crosses mentioned above (hsp70::Gal4 to UAS:myc-notch1a-intra and hsp70::Gal4 to UAS:her4) with her5 and her6 cDNAs. Whereas transcription of her5 is strongly repressed in embryos from both crosses (Fig. 8E,F), transcription of her6 seems not to be affected at all (not shown). Therefore, the fact that is a member of the hairy/E(spl) family does not necessarily imply that it is activated as a result of Notch signalling.
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Discussion |
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Her3 is an E(spl) homologue
The first conclusion is based on structural and functional considerations.
Structurally Her3 shows considerable sequence identity to proteins of the
E(spl) family in its bHLH domain, the region that binds DNA and is involved in
target recognition (Akazawa et al.,
1992; Tietze et al.,
1992
; Oellers et al.,
1994
). Furthermore, Her3 also exhibits the other characteristics
of members of this family, such as the C-terminal tetrapeptide WRPW and the
orange domain (Dawson et al.,
1995
), which corresponds to helix III/IV defined by Knust et al.
(Knust et al., 1992
). Davis
and Turner (Davis and Turner,
2001
) classify the hairy-E(spl) proteins, on structural grounds,
into four different groups. In their phylogenetic tree, Her3 belongs to the
group of E(spl) proteins. With respect to functional criteria, the effects of
fusions to the transactivation domain of VP16 and the repression domain of
Engrailed indicate that Her3 is a transcriptional repressor. Gel retardation
assays and deletion analyses support the contention that Her3 represses
transcription by binding directly to so-called N-boxes, a major DNA target for
the E(spl) proteins (Sasai et al.,
1992
; Tietze et al.,
1992
; Oellers et al.,
1994
) (for a review, see Davis
and Turner, 2001
).
The variant her3WRPW, which encodes a
Her3 derivative that lacks the C-terminal tetrapeptide WRPW, reveals an
additional element of functional similarity to E(spl). In Drosophila,
the WRPW motif is essential for the association of hairy-E(spl) proteins with
the co-repressor groucho (Wainwright and
Ish-Horowicz, 1992
; Paroush et
al., 1994
; Dawson et al.,
1995
; Fisher et al.,
1996
; Giebel and
Campos-Ortega, 1997
), and its removal results in a non-functional
polypeptide. Similarly, injections of
her3
WRPW mRNA show that this variant has
partially lost the ability to suppress target gene expression. Similar results
have been reported for her5, another member of the same protein
family (Geling et al., 2003
).
However, the WRPW tetrapeptide appears to be functionally dispensable in the
case of other members of the family. Thus, a Her4 variant lacking the WRPW
domain was found to behave like the wild-type protein
(Takke et al., 1999
).
Finally, we find that her3orange and
her3
C, which encode products that are
devoid of the orange domain and of the region between the orange domain and
the WRPW motif, respectively, behave like gain-of-function mutants. This
conclusion is based on the fact that their expression leads to a more
pronounced reduction in neurog1 transcription than does the wild-type
Her3. The deletion derivative encoded by
her3
C is similar to the product of the
E(spl)D allele of Drosophila
(Knust et al., 1987
;
Klämbt et al., 1989
),
with the exception of the WRPW-coding region, which is still present in the
former and absent in the latter. The Her3
C deletion behaves
like the product of E(spl)D when the expression of this
gene is driven by Gal4 in a Gal4-UAS experiment
(Giebel and Campos-Ortega,
1997
). When expressed under the control of Gal4,
E(spl)D behaves like a dominant-negative variant. Dominant-negative
effects were interpreted as being due to inhibition of the function of the
endogenous E(spl) proteins by competitive or neutralising interactions with
the truncated proteins (Giebel and
Campos-Ortega, 1997
). As the gel shift analyses suggest that Her3
may bind to DNA as dimers or trimers (Fig.
4F), association of the endogenous proteins with those supplied
exogenously might also explain the gain-of-function and dominant-negative
effects seen with the Her3 variants. In Drosophila, deletion of
either the orange domain or the WRPW leads to strong impairment of the E(spl)
function (Wainwright and Ish-Horowitz,
1992
; Schrons et al.,
1992
; Dawson et al.,
1995
; Fisher et al.,
1996
). It is assumed that the region between the orange domain and
the WRPW motif may be required as a spacer to accommodate Groucho, so that its
removal prevents the association of the WRPW with Groucho
(Dawson et al., 1995
;
Giebel and Campos-Ortega,
1997
).
Taken together, the results described above suggest that Her3 binds
directly to DNA and acts as a transcriptional repressor. However, mechanisms
of transcriptional repression other than direct DNA binding can not be
excluded. Thus, in addition to the bHLH domain required for DNA binding, both
the orange domain and the WRPW tetrapeptide appear to play a prominent
functional role. In fact, despite the abundance of data available (see
Davis and Turner, 2001), it
remains difficult to make generalisations with regard to how E(spl) proteins
function.
neurog1 is repressed by Her3
Our present results point to the proneural gene neurog1 as one of
the targets of Her3 function. Indeed, neurog1 transcription, as well
as that of several target genes of Neurog1, is repressed following injection
of her3 mRNA. Gel retardation assays show that neurog1
repression might be due to direct binding of Her3 to N-boxes in the
neurog1 promoter. This function is clearly compatible with the known
function of members of the E(spl) family as strong suppressors of proneural
gene function. Our data do not allow us to decide whether Her3 acts on
deltaA, islet1 and elavl3 directly or via neurog1.
Injection of morpholinos, either MO her3 or 2 blocker, leads to
ectopic expression of neurog1 and subsequent induction of a number of
targets of Neurog1, as for example deltaA, deltaD, coe2 and
her4, and the ectopic induction of primary neurone development.
However, ectopic induction of neurog1 following morpholino
injections is restricted to rhombomeres 2 and 4, whereas the remaining domains
of neurog1 expression remain unaffected. As injection of
her3 mRNA affects neurog1 transcription in all its
expression domains, the relatively mild effect of morpholino injection is a
striking result. Although we do not yet have a satisfactory explanation for
this observation, two possible hypotheses can be considered. First, it is
conceivable that under normal conditions regulatory interactions between Her3
and neurog1 are restricted to the regions of rhombomeres 2 and 4 that
connects r2MN and r2SN, and r4MN and r4SN, respectively. In this case, the
remaining expression domains of her3 would not manifest regulatory
interactions with neurog1. However, in view of the complementary
nature of the transcription patterns of her3 and neurog1,
this seems rather improbable. The second hypothesis is based on functional
redundancy of the genes of the E(spl) family. There are several
examples of members of this family being expressed in overlapping domains,
both in Drosophila and in vertebrates. In Drosophila, six
out of the seven E(spl)-C genes show identical expression pattern in the
neuroectoderm (Knust et al.,
1987; Knust et al.,
1992
; Klämbt et al.,
1989
); in the zebrafish, her4 and her2 exhibit
virtually identical expression patterns in the neural plate
(Takke et al., 1999
; Takke and
Campos-Ortega, unpublished), her1 and her7 within the
presomitic mesoderm (Oates and Ho,
2002
; Gajewski et al.,
2003
), and at least one other gene of the her family
display the same expression pattern as her5 (L. Bally-Cuif, personal
communication) (Müller et al.,
1996
; Bally-Cuif et al.,
2000
). The overlap of their expression domains and their common
function explains why the genes of the Drosophila E(spl)-C show
marked functional redundancy in early neurogenesis (reviewed by
Campos-Ortega, 1993
) (see also
Delidakis et al., 1991
;
Schrons et al., 1992
). A
similar redundancy can be invoked to explain why neurog1
transcription outside the region delimited by rhombomeres 2 and 4 is not
affected, at least to levels detectable by in situ hybridisation, by
misexpression of her3. However, as RT-PCR shows a considerable
increase in the amount of neurog1 RNA
(Fig. 4G), transcription of
neurog1 might in fact be affected in all its domains, albeit either
below levels detectable by conventional in situ techniques, or with low
penetrance. We have mentioned that, in addition to the rhombencephalon,
neurog1-positive cells are occasionally seen in other regions of the
neural plate. This would require the expression of other her genes in
the her3 domains under discussion, for which there is as yet no
evidence. Therefore, for the time being our results do not allow us to decide
between the two possibilities.
her3 is repressed by Notch signalling
Despite all the similarities between Her3 and the Drosophila
E(spl) proteins, there is one important difference, which concerns the
response to signalling through Notch1a. We find that her3
transcription is repressed by Notch signalling, in clear contrast to the
behaviour of several other members of the E(spl) family, both in
Drosophila and in vertebrates, which are activated
(Jarriault et al., 1995;
Lecourtois and Schweisguth,
1995
; Tamura et al.,
1995
; Hsieh et al.,
1996
; Kopan et al.,
1996
; Wettstein et al.,
1997
; Takke et al.,
1999
; Takke and Campos-Ortega,
1999
) (for a review, see
Lewis, 1996
). This conclusion
is based on the results of three types of experiment: (1) the downregulation
of her3 transcription observed following misexpression, both by mRNA
injection and by the Gal4:UAS technique, of notch1a-intra and
her4; (2) the upregulation of her3 transcription in embryos
expressing a dominant-negative variant of DeltaD; and (3) the downregulation
of gal4 transcription in her3::gal4 embryos, in which
gal4 is driven by the regulatory region of her3. Moreover,
our results suggest that transcriptional repression of her3 is
mediated by direct binding of Her4 to the N-boxes present in the promoter of
her3. The functional significance of the Notch1a-mediated repression
of her3 remains unclear. As Her3 represses neurog1
expression, being thus required to block neurogenesis, one possibility is that
Notch signalling promotes, rather than blocks, neurogenesis. However, this
possibility requires experimental support, which, for the time being, is still
missing.
The results obtained using the Gal4-UAS technique for targeted gene
misexpression, i.e. using both UAS:notch1a-intra and
UAS:her4, suggest that Notch1a signalling suppresses transcription of
the her5 gene as well, whereas transcription of her6, yet
another zebrafish hairy-E(spl)-related gene, remains unchanged under
the same experimental conditions. Therefore, Notch1a signalling allows us to
classify the Her genes into three different groups, depending on whether
transcription is activated (her1 and her4)
(Takke and Campos-Ortega,
1999; Takke et al.,
1999
), repressed (her3 and her5; present
results) or remains unaffected (her6; present results). Whereas
members of the first and the second group repress neurogenesis in zebrafish as
well as rodents, members of the third group promote neurogenesis, at least in
mice (Hes6) (Bae et al., 2000
;
Koyano-Nakagawa et al.,
2000
).
her5 might well be a special case among the Her genes. Thus,
Geling et al. (Geling et al.,
2003) refer to unpublished data indicating that her5
within the neural plate is independent of Notch signalling in vivo. However,
we find that her5, like her3, is downregulated by
Notch1a-intra, thus suggesting that her5 can be a target of Notch
signalling under conditions of ectopic Notch expression. Altogether, these
results suggest that Notch1a-intra can repress both her3 and
her5 expression, but only her3 expression is activated by
inhibition of Notch1a-intra, supporting the contention that her3 is a
target of Notch signalling in the embryo in vivo, and hence a new player in
the regulation of neurogenesis in zebrafish.
A case of Notch-mediated repression of Hes genes has previously been
described in the mouse. Using an in vitro assay, Beatus et al.
(Beatus et al., 1999) found
that misexpression of the intracellular domain (IC) of Notch 3 represses
transcriptional activation of Hes genes mediated by the intracellular domain
of Notch1. If cells from any of a number of different lines are transfected
with Notch1 IC together with a much larger amount of Notch3 IC, Notch1
IC-mediated transcriptional activation of Hes1 and Hes5 is repressed.
Moreover, Hes5 transcription is repressed in vivo by Notch3 IC when expression
is driven by the nestin promoter.
Formally, the effect of Notch3 IC signalling on Hes5 is the same as that of
Notch1a on her3. However, the mechanism of transcriptional repression
may not be the same in each case. The promoters of both Hes1 and Hes5 contain
RBP-J [Su(H)] binding sites and can be activated by Notch1 IC. Beatus
et al. (Beatus et al., 1999
)
suggest that Notch3 IC may either compete with Notch1 IC for access to
RBP-J
; or, alternatively, as Notch3 IC cannot activate Hes genes, it
may compete with Notch 1 IC for a co-factor present in limited amounts. In the
case of her3, sequence comparisons uncover a single high-affinity
Su(H)-binding site in the 4.7 kb genomic fragment that was used here to drive
spatially regulated transcription of her3, and has been shown to
contain Notch responsive elements. Because our results suggest that Notch1a
cannot activate her3, it is not clear what role this binding site
might play. Given that we have not tested whether other Notch receptors can
activate her3, we cannot exclude the possibility that a mechanism
like the one proposed by Beatus et al.
(Beatus et al., 1999
) is
involved in the repression of her3 transcription. However, the
available data suggest that her3 repression is caused by the
activation of her4 by Notch1a signals, and by binding of the Her4
protein to the N-boxes present in the her3 promoter. This hypothesis
is supported by the observation, mentioned above, that her4
transcription is activated by Notch1a signalling probably via Su(H) binding to
several sites in the promoter. Consequently, the mechanism would be different
from that in mouse.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
* Present address: Institute of Neuroscience, University of Oregon, Eugene,
Oregon 97403, USA
Present address: ARTEMIS Pharmaceuticals GmbH, Neurather Ring 1, 51063
Köln, Germany
Present address: Centre de Biologie du Développement (UMR 5547),
Université Paul Sabatier, 118, Rte de Narbonne, 31062 Toulouse Cedex,
France
José Campos-Ortega died on 8 May 2004
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