The genes for the helix-loop-helix proteins Id6a, Id6b, Id1 and Id2 are specifically expressed in the ventral and dorsal domains of the fish developing somites
SCRIBE-INRA, Campus de Beaulieu, 35042 Rennes, France
* Author for correspondence (e-mail: rescan{at}beaulieu.rennes.inra.fr)
Accepted 12 April 2004
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Summary |
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Key words: Id, somite, helix-loop-helix protein, myogenesis, muscle differentiation, gene expression, trout
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Introduction |
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In higher vertebrates, four different Id-encoding genes have been
described: Id1 (Benezra et al.,
1990), Id2 (Sun et
al., 1991
), Id3
(Christy et al., 1991
) and
Id4 (Riechmann et al.,
1994
). The proteins encoded by these genes have a high degree of
conservation in the HLH domain but diverge almost totally outside this region.
Orthologs of these genes have been isolated in frogs and fish, indicating that
the ancestral Id-like gene duplicated and diverged early in
vertebrate evolution (Rescan,
2001
).
Expression of muscle differentiation genes begins in trout embryos from the
20-somite stage onwards (Rescan et al.,
2001; Thiébaud et al.,
2001
). At these stages, the somite size also increases, especially
in height (Bobe et al., 2000
),
suggesting that undifferentiated somitic cells that continue to proliferate
persist alongside differentiating myocytes. One of the molecular mechanisms
that maintains these somitic cells in a proliferative state could be the
expression of Id genes. To test this hypothesis, we sought to examine
the transcription of Id genes in developing somites and to compare
Id gene expression pattern with that of muscle-specific genes.
In this study, we report the characterisation of two distinct rainbow trout
(Oncorhynchus mykiss Walbaum) cDNAs that are both orthologous to
Id6 identified in zebrafish (Danio rerio;
Sawai and Campos-Ortega,
1997). In situ hybridisation on whole trout embryos shows
that these two Id6 orthologs, as well as Id1 and
Id2, which have been previously characterized
(Rescan, 1997
), are
selectively expressed in ventral and/or dorsal domains of the developing
somite. These Id-positive areas may correspond to proliferative
somitic cells involved in somite growth.
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Materials and methods |
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Histological methods
For histological examinations, embryos were dehydrated and mounted in
paraffin, and 10 µm sections were cut. Sections were counterstained with
nuclear fast red, mounted in Eukitt (WWR International SAS, Westchester, USA)
and observed using a Zeiss 47.50.57 stereo microscope.
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Results |
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Expression of the TI6a and TId6b genes
We examined the expression pattern of TId6a and TId6b on
whole trout embryos using digoxigenin-labelled riboprobes. Our observations
are based on the Ballard (1973)
development table. The expression patterns were similar using the full-length
or the 3' UTR riboprobes (not shown). TId6a and TId6b
transcripts were found to exhibit a similar expression pattern in all
developmental stages we examined. TId6a and TId6b
transcripts were first detected at stage 11 when approximately 15 somites had
been formed. Around this stage, the labelling was observed in the most rostral
part of the paraxial mesoderm, in neoformed somites as well as in the tail bud
and the dorsal domain of the neural keel
(Fig. 2A). During the
rostrocaudal wave of somite formation, TId6a and TId6b
transcripts accumulated selectively in the ventral and dorsal regions of the
somite (Figs 2B,
3A). The persistence of
TId6a and TId6b transcripts in ventral and dorsal domains of
the myotome was observed at least until stage 20, when the segmentation is
complete to the tip of the tail (Fig.
2C). Labelling for TId6a and Tid6b genes was
also evident in the neural tube (Fig.
3A), the cerebellum, the optic tectum and the telencephalon
(Fig. 4A). The lens and the
retina were both labelled for these two transcripts
(Fig. 4A).
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Expression of the TId1 gene
As for TId6a and Tid6b, TId1 was also transcribed in the
tail bud (Fig. 5A) and the
dorsal part of the neural keel. As somitogenesis proceeded, TId1 mRNA
was detected in the rostral paraxial mesoderm as well as in ventral and dorsal
parts of the neoformed somites (Figs
3B,
5A). In contrast to TId6,
TId1 expression at the periphery of the somites was rapidly downregulated
(Fig. 5A). Staining for
TId1 was also observed in the dorsal domain of the neural tube
(Fig. 3B), the cerebellum, the
optic tectum, the telencephalon, the lens and the retina
(Fig. 4B).
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Expression of the TId2 gene
In contrast to TId6a, TId6b and TId1, the TId2
transcript was not visualized in the tail bud nor in the paraxial mesoderm but
accumulated in somites that had already been formed
(Fig. 5B). Observation of both
whole-mount embryos and transverse sections indicated that the expression of
TId2 within somites was present in a narrow domain situated in the
apical zones of the somites/myotome while no labelling was evident in the
ventral domain of the somites (Figs
3C,
5B). The labelling for
TId2 appeared transient in somites and was no longer observed after
the end of the segmentation. A strong and lasting staining was detected in the
pronephros that flanked the trunk (Figs
3C,
5B). Otherwise, in the
developing brain, TId2 transcripts were found to accumulate
preferentially in telencephalon. Only the lens was labelled in the eye
rudiment (Fig. 4C).
Somitic subdomains expressing Ids correspond to regions that do not exhibit terminal differentiation
To more fully understand the function of Id genes in developing
somites, we analysed the early somitic expression of muscular markers
including troponin C (Fig. 3D),
myosins, tropomyosins, actin, desmin
(Fig. 3E), and muscular
isoforms of aldolase A, enolase and creatine kinase. We observed that all
these muscle-specific genes were initially expressed in a medial somitic
domain that is complementary to the expression domain of the Id
genes. This indicated that Id genes are mostly transcribed in areas
(i.e. ventrally and/or dorsally) where the myoblasts are not yet fully
differentiated.
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Discussion |
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Distinct and overlapping expression of the Id genes in non-muscle tissues
Our in situ studies are consistent with an important role for the
dominant negative helix-loop-helix Id proteins in the development of
non-muscle tissues in fish. A strong accumulation of Id1, Id2, Id6a
and Id6b transcripts is observed in several discrete domains of the
brain and the spinal cord as well as in the eye rudiment. These observations,
which are reminiscent of numerous data on mammals, birds and amphibians,
emphasize the functional involvement of Id proteins in regulating nervous
system and eye development (Jen et al.,
1997; Zhang et al.,
1995
; Wilson and Mohun,
1995
; Liu and Harland,
2003
; Kee and Bronner-Fraser,
2001
). Candidate regulators of neural differentiation that can
interact with Id are orthologs of achaete-scute. An examination of the
expressed sequence tags identified in the AGENAE programs reveals the
existence of such an ortholog in the trout (accession no. BX874588),
supporting the notion that an antagonism between HLH and bHLH proteins is
probably required for proper neurogenesis in the trout.
Among the four Id transcripts examined in the present study, only
TId2 was found to be expressed in the pronephros. The role of Id2 in
regulating kidney morphogenesis and homeostasis remains unclear. The
experimental inactivation of the Id2-gene locus does not lead to an
apparent alteration of kidney development
(Yokota et al., 1999).
Nevertheless, our observation emphasizing a high level of Id2
transcription in the pronephros is in agreement with the strong accumulation
of Id2 transcript reported in the developing kidney of
Xenopus (Wilson and Mohun,
1995
) and humans (Biggs et al.,
1992
) as well as in the adult kidney of trout
(Rescan, 1997
). Interestingly,
an involvement of Id2 in regulating gene transcription in the kidney is
suggested by in vitro data showing that Id2 interacts directly with
Pax-2 and Pax-8 proteins, both of which are expressed in the kidney
(Roberts et al., 2001
).
Selective expression of Id genes in subdomains of the developing somite
All four mammalian members of the Id family interact with E-proteins and
with bHLH proteins of the MyoD family, disrupting their transcriptional
activity. Thus, it has been proposed that members of the Id family play a
regulatory role during myogenesis in mammals
(Kadesch, 1993). In the
present study, we show that the transcription of fish Id genes occurs
in forming somites at stages where myogenic regulator factors (MRFs) are
expressed (Delalande and Rescan,
1999
) and starts well before the activation of muscle structural
genes (Rescan et al., 2001
).
This raises the possibility that Id paralogs impose temporal and
spatial limits on bHLH myogenic regulator activity in fish embryos, leading to
a delay in muscle differentiation. Supporting this view, Sawai and
Campos-Ortega (1997
) have
shown that zebrafish Id6 protein antagonizes bHLH heterodimer function in
vitro. On the other hand, Id paralogs may also promote cell
proliferation in the somite subdomains, where they are expressed by
interacting with key regulators of the cell cycle
(Ruzinova and Benezra,
2003
).
Somitic transcription seems to be an ancient and conserved feature of
Id genes; indeed, the presence of Id transcripts in somites
has been observed in different phyla including not only lower and higher
vertebrates but also primitive chordates such as Amphioxus
(Meulemans et al., 2003). In
addition, it is interesting to note that Id6a, Id6b, Id1 and
Id2 expression in dorsal and/or ventral domains of the somite is
reminiscent of that of Id2, Id3 and Id4 in developing
somites of Xenopus embryos (Zhang
et al., 1995
; Wilson and
Mohun, 1995
; Liu and Harland,
2003
). This emphasizes the conservative aspects of the
transcriptional network that regulates muscle growth pattern in lower
vertebrates. Although Id1, Id2, Id6a and Id6b genes are all
restrictedly transcribed in most dorsal and ventral parts of the myotomes,
there are some differences in the temporal expression of these genes, the two
Id6 genes being the only ones that display a transcription after the
completion of segmentation. This suggests that the consequences of dominant
negative regulation of transcription factor activity within somitic cells may
be different for the different Id paralogs.
In situ hybridisation of transcripts encoding myosin heavy chains
(MyHC; Rescan et al., 2001) or
other muscle-specific proteins (present study) shows that the activation of
genes involved in muscular differentiation always starts in the medial domain
of the somite before spreading from the inside to the outside. Such an
expression pattern is complementary to that of Id1, Id2, Id6a and
Id6b. Thus, it is likely that the regionalized Id expression
in the lateral domains of the somite accounts at least in part for the delay
in muscular differentiation observed at this level. In keeping with the
regulation of Id expression in the developing myotome, it is worth
mentioning that Id expression is upregulated in vitro by
bone morphogenetic proteins (BMPs;
Hollnagel et al., 1999
), so it
would be of interest to examine whether the expression of BMPs overlaps with
that of Ids in developing trout somites. In this regard, it is
interesting to note that a BMP-like signal restricted to dorsal and ventral
regions of the fish somite would be consistent with the somite patterning
model involving opposing actions of lateral BMPs and axial hedgehogs
(Du et al., 1997
).
Bobe et al. (2000) have
observed, using scanning electron microscopy, that somite size increases,
especially in height, as soon as they form. In the light of the work presented
here, it is tempting to speculate that the germinative domains involved in
somite growth are situated in the Id-positive ventral and dorsal
regions that are the last to differentiate. Such a growth pattern involving
ventral and dorsal subdomains of the somite raises the possibility that a
growth process similar to the stratified hyperplasia observed in late embryos
and in larvae (Rowlerson and Veggetti,
2001
) may occur as soon as the somite forms.
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Acknowledgments |
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