1 Zebrafish Neurogenetics Junior Research Group, Institute of Virology,
Technical University-Munich, Trogerstrasse 4b, D-81675 Munich, Germany
2 GSF-National Research Center for Environment and Health, Institute of
Developmental Genetics, Ingolstaedter Landstrasse 1, D-85764 Neuherberg,
Germany
3 Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235
USA
Authors for correspondence (e-mail:
ninkovic{at}gsf.de
and
bally{at}gsf.de)
Accepted 6 October 2004
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SUMMARY |
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Key words: Hairy, E(spl), her5, him, Midbrain-hindbrain, MHB, Neurogenesis, Zebrafish
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Introduction |
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The embryonic midbrain-hindbrain domain (MH) is characterized by the
maintenance of a zone of delayed differentiation at the midbrain-hindbrain
boundary (MHB). This zone, called `intervening zone' (IZ), separates midbrain
from anterior hindbrain neuronal clusters and has been described in all
vertebrates (Bally-Cuif et al.,
1993; Geling et al.,
2003
; Vaage,
1969
). Its functional importance is attested by genetic ablation
experiments. For instance, in mouse embryos lacking the function of the two
bHLH E(spl)-like transcription factors Hes1 and Hes3, premature
differentiation of the IZ occurs, leading to the lack of several MH neuronal
populations, and to the collapse of MH structures
(Hirata et al., 2001
). These
defects may primarily result from a disruption of the isthmic organizer, an
inducing cell population located at the MHB and involved in MH maintenance
(Hirata et al., 2001
;
Martinez, 2001
;
Rhinn and Brand, 2001
;
Wurst and Bally-Cuif, 2001
).
Recent results in zebrafish have permitted dissection of the mechanisms of IZ
formation in more detail. There, expression of the hairy/E(spl) gene
her5 (Muller et al.,
1996
) precisely delineates the IZ at all embryonic stages
(Geling et al., 2003
). At the
onset of neurogenesis (tail-bud stage), her5 expression separates the
early midbrain ventrocaudal proneural cluster (vcc) from the anterior
hindbrain proneural clusters of rhombomere 2 (presumptive motorneurons -r2MN-
and lateral neurons -r2LN-). In the absence of Her5 function, ectopic
neurogenesis occurs in the medial (future basal) part of the IZ, as revealed
by the ectopic expression of the proneural genes neurogenin1
(ngn1) and coe2 and the later differentiation of neurons
across the basal MHB, bridging the vcc and r2MN
(Geling et al., 2003
;
Geling et al., 2004
).
Conversely, forced expression of ngn1 within the MH domain leads to a
partial downregulation of MHB markers' expression
(Geling et al., 2003
). These
results have two implications. First, they confirm that the IZ is necessary to
maintain MHB integrity. Second, they demonstrate that the IZ is composed of at
least two domains along the mediolateral axis, which differ in their
requirement for Her5 function: the medial IZ domain (MIZ), which crucially
depends on Her5 for neurogenesis inhibition, and the lateral (future alar) IZ
domain (LIZ), which forms even in the absence of Her5. Within the MIZ, Her5
acts as a prepattern factor that prevents the formation of a proneural
cluster, and inhibits expression of ngn1 and coe2 upstream
of Notch signaling (Geling et al.,
2004
).
Our study of Her5 function did not address the formation of the LIZ, in
spite of its crucial role in controlling midbrain and anterior hindbrain alar
neurogenesis. To now approach this issue, we reasoned that other Hairy/E(spl)
factors might be expressed within this domain and act redundantly with Her5.
Because there are examples of physically linked E(spl) genes in
Drosophila (E(spl) complex)
(Klambt et al., 1989;
Knust et al., 1992
) and
zebrafish (her1 and her7)
(Henry et al., 2002
), and
because linked genes are more likely to share spatiotemporal characteristics
of expression, we searched for new Hairy/E(spl) genes in the vicinity
of the her5 locus. Sequencing a her5-containing PAC revealed
a new her-like gene, him, adjacent to her5 and in
opposite orientation (hence him for her5
image), identically expressed across the IZ. We report here that
Him is the hypothetical factor cooperating with Her5 to control LIZ formation
in vivo, and that Him also crucially contributes to MIZ formation. Together,
our results unravel the genetic combination preventing neurogenesis across the
MHB.
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Materials and methods |
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The phylogenetic tree of zebrafish Her family was done using the phylodendrone software (www.es.embnet.org/Doc/phylodendron/treeprint-form.html). To construct the phylogenetic tree, the VectorNTI software and full-length sequences were used. The accession numbers of compared proteins are: Her5: NP_571152, Her1: NP_571153, Her7: NP_571684, Her4: NP_571165, Her2: NP_571164, Her3: NP_571155, Her9: NP_571948 and Her6: NP_571154. Proteins predicted by The Wellcome Trust Sanger Institute are: Her11 (ENSDARP00000012990), Her13 (ENSDARP0000008307), Hes6 (ENSDARP00000021078) and Her12 (ENSDARP00000038100) (http:www.ensembl.org, release from 08-02-2004).
Zebrafish strains and transgenic lines
Embryos obtained from natural spawning of AB wild-type or transgenic fish,
her5PAC::egfp (Tallafuss and Bally-Cuif,
2003) and -8.4ngn1::egfp
(Blader et al., 2003
), were
raised and staged according to Kimmel et al.
(Kimmel et al., 1995
). no
isthmus (noitu29a), acerebellar
(aceti282a) (Brand et
al., 1996
) and knypek (b404, knym119)
(Solnica-Krezel et al., 1996
;
Topczewski et al., 2001
)
mutants were obtained by pairwise mating of heterozygous adult carriers, as
described previously.
Protein expression interference assays
Morpholino antisense oligonucleotides (her5MOATG) were
purchased from Gene-Tools, Inc. (Oregon, USA). The morpholino was dissolved to
a stock concentration of 5 mM in H2O and injected into one-cell
stage embryos at 1 mM. himMOs lead to non-specific cell death (not
shown). Thus, GripNA antisense oligonucleotides preventing
him translation, specific for the him ATG region
(himGripNAATG) or acceptor site of the second him
exon (himGripNASPL), were purchased from Active Motif
(Belgium). GripNAs were dissolved to stock solution of 1 mM in
H2O and injected into one-cell stage embryos at 0.5 mM. At this
dose, the effect of himGripNAs on ngn1 expression was
maximal. Sequences of antisence oligonucleotides were as follows:
her5MOATG: 5'-TTGGTTCGCTCATTTTGTGTATTCC-3';
himGripNAATG: 5'-ATTCGGTGTGCTCTTCAT-3' and
himGripNASPL: 5'-TACTCACAGTGTCTGCAG-3'. All
injection experiments were repeated at least three times.
In situ hybridization and immunohistochemistry
Probe synthesis, in situ hybridization and immunohistochemistry were
carried out as previously described
(Hammerschmidt et al., 1996).
The following in situ antisense RNA probes were used: her5
(Muller et al., 1996
),
him (this paper), ngn1
(Korzh et al., 1998
),
pax2.1 (Lun and Brand,
1998
) and egfp (Clontech). Primary antibodies used for
immunohistochemistry were rabbit anti-GFP (ams biotechnology Europe, TP401)
used at a final dilution of 1/500 and mouse anti-human neural protein HuC/HuD
(MoBiTec A-21271) (1/300). They were revealed by using FITC-conjugated goat
anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories,
111-095-003) or Cy3-conjugated goat anti-mouse secondary antibody (Jackson
ImmunoResearch Laboratories, 115-165-044) (1/200), as appropriate. Embryos
were scored and photographed under a Zeiss SV 11 stereomicroscope or a Zeiss
Axioplan photomicroscope.
RNA injections
knypek capped RNA was synthesized using Ambion mMessage mMachine
kit following the recommended procedure. Capped RNA was injected at the
concentration of 60 ng/µl into the embryos at the one-cell stage.
Protein interaction assays
For two-hybrid assays, The MATCHMAKER GAL4 Two-Hybrid System 3 (Clontech)
was used following procedures described by the manufacturer. The `bait' and
`AD' plasmids were constructed by fusing in-frame the complete ORFs of
her5, him and ngn1 to either pGBKT7 (encoding the GAL4
DNA-binding domain) or pGADT7 (encoding the GAL4 activation domain). The
relative stringency of Her5 homodimerization versus its heterodimerization
with Him was quantified by ß-galactosidase assay according to the
manufacturer's recommendation (Clontech). The ß-galactosidase activity
was quantified according to Lazo et al.
(Lazo et al., 1978).
Co-immunoprecipitation and western blot analysis
Transformed yeast cells expressing the two proteins of interest were lysed
in 0.5 ml of lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.1% NP40, 0.1 mM
DTT, 0.1 mg/ml pepstatin A, 0.03 mM leupeptin, 145 mM benzamidine, 0.37 mg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride) with 0.25 g of glass beads
(425-600 µl, Sigma) for one hour at 4°C with shaking. The extracts were
then centrifuged at 15 000 g for 10 minutes at 4°C to
eliminate cell debris, and the supernatant was collected. For each
immunoprecipitation, 0.4 ml aliquots of lysate were precleaned by incubation
with 150 µl of pre-immune rabbit serum and 100 µl of 1:1 slurry of
Protein A SepharoseTM CL-4B (Amersham Bioscience AB) for 30 minutes at
4°C. Precleaned extracts were immunoprecipitated with 10 µl of rabbit
anti-HA antibody (dilution 1/1000) (Sigma) and 100 µl of 1:1 slurry of
Protein A Sepharose CL-4B (Amersham Bioscience AB) for 30 minutes at 4°C.
The sepharose beads were washed three times with 1 ml of lysis buffer. The
precipitates were fractionated on SDS-PAGE and subsequent western blot
analysis was performed according to standard protocols, by using mouse
anti-c-Myc antibodies (1/1000) (Sigma). The primary antibodies were revealed
using HRP-coupled secondary antibodies (Jackson Laboratories) diluted to 1/200
and enhanced chemiluminescence (Amersham Bioscience AB).
Quantification of him mRNA in the her5PAC::egfp line
Total RNA was isolated from her5PAC::egfp embryos and WT
siblings at the five-somite stage and reverse-transcribed before real-time PCR
amplification. Real-time PCR was done by using LightCycler FastStart DNA
Master SYBR Green I kit (Roche, Germany) and Light Cycler Instrument (Roche,
Germany). Quantitative values were obtained from the threshold cycle number at
which the increase in the signal associated with exponential growth of the PCR
products begins to be detected using the LightCycler Software, according to
the manufacturer's recommendations. The precise amount of total RNA added to
each reaction mix (based on optical density) and its quality (lack of
extensive degradation) are both difficult to assess precisely. We therefore
also quantified the transcript of the pax6 gene as the endogenous RNA
control, and both samples were normalized to the basis of pax6
content (R-value on the graph). The nucleotide sequences of the
specific primers used are shown in Table
1. The thermal cycling conditions comprised an initial
denaturation step at 95°C for 10 minutes and 65 cycles at 95°C for 15
seconds, 55°C for 10 seconds and 72°C for 15 seconds. The
quantifications were performed in triplicate on a pool of 50 embryos for each
line and results represent the mean value±s.e.m.
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Results |
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The Him protein, translated from our full-length cDNA sequence, consists of
297 amino acids and exhibits all structural features of an Hairy/E(Spl) bHLH
factor acting as transcriptional repressor
(Davis and Turner, 2001): a
conserved proline residue in the basic domain, an `orange' domain
(Dawson et al., 1995
) and a
WRPW tetrapeptide in the C-terminus
(Fisher et al., 1996
)
(Fig. 1C). Within the zebrafish
Her family, Him shows the highest similarity to Her1 with 28.6% identical and
35.2% conserved amino acid (aa) residues. Similarity between Him and Her5 is
slightly weaker (20.1% identical and 29.2% conserved aa residues)
(Fig. 1B, red lines, and
Fig. 1C). Comparison restricted
to the functional bHLH domain reveals 66% identity to Her1 and 50% identity to
Her5.
him expression within the presumptive midbrain-hindbrain is identical to her5 and marks the intervening zone
We analyzed him expression by RT-PCR and in situ hybridization.
him, like her5, is maternally expressed
(Fig. 2A). Early zygotic
him expression is ubiquitous (data not shown) but rapidly resolves in
a first, transient, profile within the presumptive dorsal endoderm and
mesoderm (Fig. 2B) at 30%
epiboly: him is expressed in deep scattered cells of the dorsal
embryonic margin and in the deep layer of the dorsal mesendoderm
(Fig. 2B and 2B', red
arrows). From mid-gastrulation onwards (70% epiboly), him expression
in the presumptive endo- and mesoderm becomes undetectable
(Fig. 2C). At that stage,
him becomes transcribed in the anterior neural plate, in a V-shaped
domain interrupted at the midline (Fig.
2C, red arrowhead). Expression in the lateral aspects of this
domain is slightly broader and stronger than medially. At the three-somite
stage, this expression fuses medially and, by anatomical landmarks, is clearly
located within the presumptive MH domain
(Fig. 2E,F). him
expression is maintained at the MHB later on until 36 hpf
(Fig. 2H,I). Starting at late
gastrulation, him is also expressed in the presomitic mesoderm
(Fig. 2D-F, blue arrows).
Expression in this territory is detectable until late somitogenesis
(Fig. 2H).
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Taken together, our data suggest a more complex picture than expected from the genomic organization of him and her5. him and her5 share expression within the MH domain, but differ elsewhere in her5-specific versus him-specific domains (pharyngeal precursors versus presomitic mesoderm at late gastrulation, respectively). In the neural plate however, Him is, together with Her5, the earliest marker of the MH and IZ, prompting us to analyze its function in this domain in more detail, in relation to MH patterning and Her5 activity.
Like Her5, and in contrast to most MH factors, Him does not control patterning events within the MH region
Refined regionalization and maintenance of the MH domain at somitogenesis
stages depends on a positive cross-regulatory loop involving Fgf8 and Pax2.1
(Brand et al., 1996;
Lun and Brand, 1998
;
Reifers et al., 1998
;
Tallafuss and Bally-Cuif,
2003
). To determine whether him was part of this loop, we
analyzed its expression in ace/fgf8 and noi/pax2.1 mutants
(Brand et al., 1996
;
Lun and Brand, 1998
;
Reifers et al., 1998
).
Expression of him in ace mutant embryos is initiated
normally (data not shown) but, from mid-somitogenesis stages onwards,
gradually narrows to persist at the MHB only in a dorsal patch
(Fig. 3A,B). At 24 hpf
him expression in ace is undetectable (data not shown).
Similarly, in noi mutants, a downregulation of him
expression can be observed from mid-segmentation stages onwards. In contrast
to ace, him expression in noi later remains restricted to
the ventral MHB (Fig. 3C,D),
like her5 (Lun and Brand,
1998
; Reifers et al.,
1998
). Together, these observations demonstrate that the
maintenance of him expression is, like that of other MH genes and
within a similar time-window, under control of the MH regulatory loop.
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Thus, although him expression depends on the MH maintenance loop, Him activity itself does not appear to impinge on this loop to influence MH patterning, like that of Her5.
Him activity is crucial for the formation of the medial IZ at early neurogenesis stages
We demonstrated previously that Her5 is crucially necessary to prevent
neurogenesis across the MIZ (Geling et
al., 2003). Because of the similar expression of him, and
its encoding a related Hairy/E(Spl) factor, we explored a potential
involvement of Him in IZ formation. At early neurogenesis stages, the IZ is
also the major site of pax2.1 expression, which we used as landmark
in future experiments (see Fig.
4D).
|
Him and Her5 are independently required for medial IZ formation
The above results are compatible with a simple model where Him and/or Her5
would act in a common regulatory cascade, one factor positively regulating
expression of the other gene. Thus, loss of Him function would cause loss of
her5 expression, or the reverse. Alternatively, Him and Her5 might be
independently necessary for MIZ formation. To address this question, we
studied him and her5 expression in embryos where Her5 or Him
activity, respectively, was blocked. We observed that him expression
was unchanged in Her5 morphants, in both the MH region and the presomitic
mesoderm, under conditions where ngn1 expression was strongly induced
in place of the MIZ (Fig. 4E,F,
and data not shown). Thus him expression is not under immediate
control of Her5. Likewise, injection of GripNAATG into
wild-type embryos did not produce alterations in her5 transcription,
although the MIZ was lost (Fig.
4I compared to
4K, and data not shown).
Furthermore, injection of GripNAATG did not affect the
production of the fusion Her5-GFP protein, driven under control of all
her5 regulatory elements in her5PAC::egfp transgenics
(Tallafuss and Bally-Cuif,
2003) (Fig. 4J,L).
Thus, Him does not influence her5 transcription or translation.
We conclude that Him and Her5 do not act in a simple cascade of cross-regulation of expression. Rather, the two genes are expressed independently of each other and are both essential to MIZ formation.
The crucial determinant of MIZ formation is the total dose of Him + Her5 inhibitory activities
Several hypotheses could account for the above results. First, Him and Her5
might both be required for MIZ formation because they need to heterodimerize
with each other to be active. Alternatively, these factors do not have unique
essential activities, but rather are required to reach together a threshold
level of Hairy/E(spl) activity necessary to prevent proneural gene expression.
Finally, both factors might exert distinct and/or complementary functions
necessary for MIZ formation.
To unravel the relevance of each hypothesis in vivo, we first tested
whether Him and Her5 could interact in a yeast two-hybrid system. bHLH factors
have the capacity to dimerize via their HLH domain, however their affinity for
hetero- versus homodimerization cannot be a priori predicted, and some
instances of DNA binding as oligomers have also been reported
(Firulli et al., 2000;
Iso et al., 2001
;
Wainwright and Ish-Horowicz,
1992
). We observed that Her5 can homodimerize as well as bind
Ngn1, while Him and Ngn1 failed to interact. In addition, heterodimers of Him
and Her5 were produced. Whether Him is also able to homodimerize could not be
tested due to unexplained toxicity of the him-expressing constructs
(Table 2). All these
interactions were confirmed by coimmunoprecipitation
(Fig. 5A). Moreover, the
affinity for heterodimerization between Him and Her5, based on
beta-galactosidase activity (Lazo et al.,
1978
), appeared six-fold higher than the affinity of Her5 for
homodimerization (Fig. 5B),
suggesting that the Him-Her5 configuration predominates in vivo if the amount
of proteins is equal. Thus, the requirement for Him and Her5 for MIZ formation
in vivo might indeed be explained by the necessity for these factors to
heterodimerize.
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We conclude from these observations that the crucial component of MIZ formation and maintenance is a threshold level of `Him + Her5' inhibitory activity. In the normal embryo, this level is probably achieved by Her5-Him heterodimers, although a possible contribution of homodimers and/or oligomers from each factor separately cannot be excluded.
Formation of the lateral IZ also relies on the level of `Him + Her5 activity' but with a lower threshold than the medial IZ
The LIZ is preserved in both her5 and him single
knockdown embryos, suggesting that it might require other factors than Him and
Her5 for its formation. Alternatively, the LIZ might primarily differ from the
MIZ in requiring a lower threshold of `Him + Her5' activity, the endogenous
level of one factor alone (two doses) being sufficient to block neurogenesis
in this location. To address these hypotheses we assayed for lateral
ngn1 expression in double knockdown embryos obtained by the
co-injection of her5MOATG and
himGripNAATG. Strikingly, we observed that the
simultaneous interference with both Her5 and Him activities results in ectopic
ngn1 expression in place of the entire IZ, i.e. including the LIZ
(88% of cases, n=25) (Fig.
6A,A' compared to
6D,D'), in striking
contrast to single knockdowns (0% of cases for her5 knockdowns,
n=21, 0% of cases for him knockdowns, n=24)
(Fig. 6B,C).
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Him and Her5 are equally potent neurogenesis inhibitors in the lateral IZ
We demonstrated above that the total amount of Him + Her5 inhibitory
activity is the crucial determinant for IZ formation, and that increased
levels of Him could compensate for loss of Her5 both in the medial and lateral
IZ. These experiments however did not address the relative contribution of
Her5 to the total inhibitory activity required for IZ formation. To determine
whether Him and Her5 contribute equally to this activity, we analyzed LIZ
formation in b404/+; knym119/+ embryos where the
function of either Her5 (Fig.
7K) or Him (Fig.
7L) was abolished. This background, obtained by crossing
b404/+ with knym119/+ heterozygote adults, allows
immediate identification of the embryos carrying one single copy of each gene
him and her5, since such embryos display the knypek
phenotype. Blocking the activity of Him or Her5 b404/+;
knym119/+ embryos leaves only one functional copy of
either her5 or him, respectively. Assaying for ngn1
expression revealed that the LIZ forms normally in such embryos (Fig.
7K,K' and
7L,L' compared to
7M,M', white arrowhead)
(while, as expected, the MIZ is lost, Fig.
7K, and
7L, compared to
7M, white arrow). These results
are in agreement with an equal potency of Him and Her5 to inhibit lateral
neurogenesis at the MHB and, like for the MIZ, we propose that a crucial
determinant of LIZ formation is the threshold of Him + Her5 activities rather
than the specific presence of both factors.
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Discussion |
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him and her5 are a new co-functional gene pair
him and her5 are located 3 kb apart in a head-to-head, in
a manner reminiscent of the her7/her1 gene pair
(Henry et al., 2002). Our
search through the zebrafish genome revealed an additional similarly organized
pair of her genes, her4/her12, located on
chromosome fragment ctg10516 ESTs BM023698 and AL716753 match with 100% to
the cDNA sequence deducted from ENSDARG00000028110, suggesting that
her12 is a real transcribed gene. Thus, our results suggest that a
paired and divergently transcribed configuration is a frequent organization of
zebrafish her genes. Our search through other vertebrate genomes,
however, including mouse and Fugu rubripes (Fugu genome, The
Welcome Trust Sanger Institute, release 08-02-04), failed to reveal a similar
organization of hairy/E(spl)-like genes in these species, suggesting
that the molecular process(es) generating her pairs took place along
the lineage leading to zebrafish.
Our results do not suggest a simple evolutionary model leading to the
generation of zebrafish her pairs. Indeed, the six her genes
involved belong in sequence to two groups of orthologs (group 1:
him/her5/her1 versus group 2: her7/her4/her12), but only the
her7/her1 pair contains one gene from each group. Thus the situation
is not comparable to dlx gene pairs, interpreted to result from a
tandem duplication followed by a cluster duplication. Because her4
and her12 have very similar coding sequences, it is possible that
these two genes underwent a recent event of gene conversion, facilitated by
the formation of intrachromosomal hairpins
(Hickey et al., 1991). We
failed to detect indications supporting gene conversion within the
him/her5 gene pair, but other recombination events might have
occurred (D. Chourrout, J.N. and L.B.-C., unpublished observations).
Linked genes sharing sequence similarity have been reported for a variety
of genetic functions in several organisms.
(Akam, 1989;
Alonso and Cabrera, 1988
;
Bober et al., 1994
;
Coleman et al., 1987
;
Kmita and Duboule, 2003
;
Knust et al., 1992
;
Stein et al., 1996
).
Duplication events resulting in linked arrays of related genes generate copies
that often share cis-acting regulatory sequences. Whether him and
her5 expression across the IZ are coregulated remains to be directly
demonstrated but is highly likely, given that the enhancer driving MH
expression of her5 extends into the him locus
(Tallafuss and Bally-Cuif,
2003
). In addition, him and her5 differ in some
aspects of their expression profiles (in the shield and presomitic mesoderm,
versus presumptive pharyngeal endoderm, respectively). The regulatory elements
controlling endodermal expression of her5 are located closer to the
her5 ATG than the MH expression elements
(Tallafuss and Bally-Cuif,
2003
). Thus expression of the him/her5 pair may be
controlled by a combination of proximal and gene-specific elements (accounting
for the differential expression sites of the two genes) and distal and
probably common elements (driving IZ expression). It is possible that the
proximal elements are new modifications in the evolution of the gene pair,
extending genetic functions by the acquisition of new expression domains
(Ohta, 2000
). It will be
interesting to determine whether such cis-regulatory organization is involved
in generating different expression sites within other gene pairs.
The combined activities of Her5 and Him determine LIZ formation
We previously identified Her5 as the first determinant of MIZ formation in
zebrafish (Geling et al.,
2003). However, although her5 expression covers the whole
IZ, and ectopic her5 expression can inhibit ngn1 in the
lateral MH area, we failed to implicate Her5 alone in LIZ formation in vivo. A
main advance of our present work is to provide an interpretation for this
finding, by identifying a new Hairy/E(spl) factor, Him, as the partner for
Her5 in LIZ formation. Our arguments rely on the phenotype of embryos where
the functions of Her5 and Him are concomitantly blocked in a non-genetic
interference approach, and of embryos carrying the b404 deletion,
where both him and her5 genes are absent. In both cases,
ectopic ngn1-positive cells replace the LIZ. This phenotype is not
found by blocking the function of either Him or Her5 alone, and is rescued by
selectively reintroducing endogenous levels and profile of Him function into
the b404 background, arguing for its specificity. Further, we show
that one copy of either him or her5 (as in
b404/+;knym119/+ heterozygote embryos where Her5
or Him function is blocked) similarly preserves the LIZ, demonstrating that
Him and Her5 are equally potent at inhibiting ngn1 expression in that
location. Thus, our results identify Him and Her5 as truly redundant factors
that play equivalent roles, and are together the only determinant, in LIZ
formation.
The molecular cascade downstream of Him remains unknown. Because Him is
sufficient for LIZ formation in the absence of Her5, we can exclude a
mechanism where Him would primarily promote Her5 activity. Rather, because of
the similar sequences of Him and Her5, it is more likely that both factors act
together on common targets controlling neurogenesis. Within the MIZ, Her5 and
Him (J.N. and L.B.C., unpublished) act upstream of Notch to inhibit expression
of ngn1 and coe2, but not other early MH proneural genes
such as asha, ashb and ato3
(Geling et al., 2003). It will
be important to determine whether the molecular cascade(s) and mechanisms
downstream of Her5 and Him are conserved in the MIZ and LIZ. Compared to the
MIZ, the LIZ exhibits an additional block, still molecularly unknown, that
prevents neurogenesis downstream of Ngn1 activity
(Geling et al., 2003
). Whether
Him and Her5 also take part in this second block remains to be tested.
A unified model for IZ formation along the entire mediolateral extent of the neural plate
The absence of Her5 leads to disappearance of the entire MIZ and its
replacement by ngn1-expressing cells, which later differentiate into
Hu-, HNK1- and acetylated-tubulin-positive neurons
(Geling et al., 2003).
Surprisingly, our results now demonstrate that Him plays an equally important
role in MIZ formation, since an exactly identical phenotype is triggered by
lack of Him activity (this paper, and data not shown). We further rule out an
interdependent regulation of him and her5 expression
(Fig. 4E-L). Thus, another
important implication of our work is that MIZ formation relies on
prepatterning by both Him and Her5.
A priori, the finding that loss of Him or Her5 function result in identical phenotypes can have three different molecular interpretations: first, Him and Her5 might act in distinct pathways that converge on and are both necessary for neurogenesis control at the MIZ; second, the activities of Him and Her5 might be interdependent; third, Him and Her5 might have equivalent functions, a minimal dose of `Him + Her5' activity being required for MIZ formation. The first two mechanisms are unlikely, given the observation that increased levels of Him alone to three doses (as in her5PAC::egfp/+ heterozygote transgenic embryos injected with her5MO, Fig. 5E) can compensate for the lack of Her5 function within the MIZ. We do not have genetic means of assessing whether a high dose of Her5 alone would also suffice for MIZ formation. However, our findings that Him and Her5 are equally potent to prevent lateral neurogenesis strongly suggest that this is the case. Thus, we propose that the crucial determinant of MIZ formation, is a total level of `Him + Her5' inhibitory activity. Hence, above a threshold of Him + Her5, ngn1 expression is prevented medially and the MIZ is formed, while ngn1 expression is induced below this threshold (Fig. 8). As discussed above, our results indicate that three doses of one factor alone is the minimum level of inhibitory activity required for MIZ formation. Interestingly, however, two doses are sufficient when both Him and Her5 are present, as in b404/+ heterozygote embryos. This result might be related to the higher propensity of Him and Her5 to hetero- than homodimerize, or to an increased activity of heterodimers versus homodimers or oligomers. Because the same factors Him and Her5 account for LIZ formation, and can functionally replace each other in this domain as well, a parsimonious interpretation of our findings is to implicate the same dose-dependent mechanism within the LIZ, albeit with a lower threshold level (Fig. 8). The LIZ minimal level of inhibition would be achieved with one dose of Him or Her5 alone. Together, our results thus lead to a unified model where the maintenance of a pool of progenitor cells at the MHB is orchestrated by a variable dose-dependency to the Him/Her5 pair.
|
An interesting open question remains to identify the cues controlling the
differential sensitivity of the MIZ versus LIZ to Him + Her5, and their
functional significance. The MIZ and LIZ differ in their proliferation rates:
the MIZ exhibits more cells in M phase than the LIZ at late gastrulation,
based on anti-phosphoH3 immunostaining
(Geling et al., 2003). It will
be crucial to investigate the possible relationship between MIZ and LIZ cell
cycle properties and their response to Him + Her5. Also, several morphogens
acting in this region are expressed following a mediolateral gradient. For
instance, wnt1 is expressed in a spatio-temporal pattern similar to
her5 and him at late gastrulation, thus with initially
higher levels laterally than medially, and might enhance cell sensitivity to
neurogenesis inhibitors. This might be related to the delay of dorsal
differentiation proposed to result from the gradient of Wnt signaling from the
spinal cord roof plate (Megason and
McMahon, 2002
). Conversely Shh signaling from the ventral midline
and specifically active at the MHB (Carl
and Wittbrodt, 1999
; Koster et
al., 1997
) could increase `neurogenic competence'. These
hypotheses will be important to test experimentally to gain insight into the
prepatterning of IZ formation.
Biological significance of a redundant process for IZ formation
Redundant factors are generally viewed as `safety' locks, and the
biological significance of the Him/Her5 couple might be to secure IZ
formation. This case of redundancy is more extreme than observed for
Her1/Her7, where the disruption of each gene alone produced distinct (although
moderate) somitic defects, indicating partially different activities
(Henry et al., 2002). The
embryonic MHB progenitor pool serves several vital functions. It generates the
large majority of MH neurons and glia, as demonstrated in lineage tracing
experiments (Tallafuss and Bally-Cuif,
2003
) and genetic or surgical ablation
(Cowan and Finger, 1982
;
Hirata et al., 2001
). MH
neurons form crucial integration centers involved in visual, auditory and
motor control and social behavior. By its long-lasting proliferative activity,
the IZ also permits the expansion of MH tissue over time. Although the
relative importance of MH derivatives varies between species, most vertebrates
are characterized by highly developed visual, auditory or locomotor functions,
which are paired with enlarged mesencephalic derivatives or cerebellum.
Finally, and importantly, the IZ coincides in space with the isthmic
organizer, necessary for patterning the entire MH domain and for the
subdivision of mid-versus hindbrain structures
(Bally-Cuif et al., 2000
;
Liu and Joyner, 2001
;
Rhinn and Brand, 2001
). In the
mouse, IZ formation also relies on the two redundant bHLH factors Hes1 and
Hes3 (Hirata et al., 2001
). In
that case however Hes1 and Hes3 are not genetically linked
and their expression profiles are clearly distinct, overlapping only at the
MHB (Allen and Lobe, 1999
;
Lobe, 1997
), suggesting that
mouse and zebrafish have independently evolved a strategy for the redundant
expression and function of Hairy/E(spl) factors at the MHB. A dose dependency
and the spatial details of IZ formation in the mouse have not been explored.
The fact that one dose of each factor Him and Her5 suffices to maintain the
MIZ in zebrafish, while two doses of each single factor do not, probably
explains the maintenance of the two genes him and her5 in
zebrafish. Whether Hes1 and Hes3, or Him and Her5, exert in addition other and
perhaps distinct activities at the MHB remains to be explored.
The IZ is not an isolated case of maintenance of a non-differentiation zone
at embryonic signaling boundaries. Such events have been reported, e.g. at the
Drosophila wing margin, along the dorsal and ventral midlines of the
neural tube (Alexandre and Wassef,
2003), as well as between rhombomeres
(Cheng et al., 2004
). Like the
IZ, these boundaries are involved in the progressive building and patterning
of their adjacent territories, and the maintenance of their integrity
necessitates their remaining undifferentiated. This process is achieved by
Notch signaling at the wing margin and inter-rhombomeric boundaries, and Shh
signaling along the neural tube ventral midline, while the factors involved
along the dorsal midline probably involve Wnt and BMP signaling. Our work
demonstrates that a distinct molecular mechanism accounts for
non-differentiation at the MHB, namely the differential response of MHB cells
to the combined inhibitory activity of two twin and co-regulated
Hairy/E(spl)-like factors, independently of Notch. Our findings add to the
panel of identified developmental strategies used to build and maintain
signaling centers.
Note added in proof
him is identical to her11, which has been recently
reported for its role in zebrafish somitogenesis
(Sieger et al., 2004). The
gene referred to as her11 in the present manuscript
(Fig. 1B, ENSDARP00000012990),
named before publication by Sieger et al., is a different coding sequence and
corresponds to her13 of Sieger et al. The gene referred to as
her13 in the present manuscript
(Fig. 1B, ENSDARP0000008307) is
a new gene, not reported by Sieger et al. We suggest that the latter gene be
renamed her16 and that the nomenclature of Sieger et al. be used for
all other genes in future work.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
* Present address: Institute of Neuroscience, University of Oregon, Eugene,
OR 97403, USA
Present address: Children's Memorial Institute for Education and Research,
Northwestern University Feinberg School of Medicine, Chicago, IL 60614,
USA
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