Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries, 67404 Illkirch Cedex, CU de Strasbourg, France
Author for correspondence (e-mail:
uwe.straehle{at}itg.fzk.de)
Accepted 15 September 2004
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SUMMARY |
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Key words: neurogenin1, Pax6, Telencephalon, Diencephalon, Evolution, Neurogenesis, Transcription, Regulatory elements, Co-option, Zebrafish, Mouse
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Introduction |
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Central, but less well understood questions concern how the spatial aspects
of neurogenesis are controlled in the vertebrate neural plate/tube and how
these mechanisms have been modified during vertebrate evolution. Neurogenins
(ngns), which belong to the bHLH family and which are most closely related to
the Drosophila bHLH factors Biparous and Atonal, demarcate the
regions of primary neurogenesis in the neural plate of zebrafish and
Xenopus embryos (Blader et al.,
1997; Ma et al.,
1996
). Accumulating evidence suggests that these
ngn1-expressing regions in the neural plate of lower vertebrates are
specified by pre-pattern genes that provide positional codes with their large
and overlapping domains of expression
(Bally-Cuif and Hammerschmidt,
2003
). Genes of the iroquois family, members of which are
involved in positioning the expression of the proneural genes achaete
and scute in the Drosophila imaginal disc, have been
suggested to play similar roles in the vertebrate neural plate
(Bellefroid et al., 1998
;
Cheng et al.,2001
;
Gomez-Skarmeta et al., 1998
;
Itoh et al., 2002
;
Wang et al., 2001
). In
Xenopus embryos, the zinc-finger transcription factor Zic2 acts as a
repressor of ngn1 expression in the longitudinal stripes, separating
the ngn1-expressing precursors of motor neurones, interneurones and
sensory neurones (Brewster et al.,
1998
). Another negatively acting factor, the bHLH factor Her5,
prevents neurogenesis at the midbrain/hindbrain boundary (MHB). Inhibition of
Her5 function leads to an expansion of ngn1 expression and ectopic
formation of a proneural field in the MHB area
(Geling et al., 2003
).
In the neural tube of mouse embryos, the paired-homeodomain transcription
factor Pax6 has important regulatory functions
(Ashery-Padan and Gruss, 2001;
Simpson and Price, 2002
). Pax6
mutant mice develop small eyes and have deficiency in neurogenesis in the
brain and spinal cord. In both the telencephalon and the spinal cord, Pax6 is
expressed in a graded fashion, suggesting that it provides
concentration-dependent positional information for the region-specific
differentiation of neural tissues
(Stoykova et al., 2000
;
Scardigli et al., 2003
). The
zebrafish genome encodes two pax6-related genes, pax6.1 and
pax6.2, that are expressed in overlapping domains in the eye, dorsal
diencephalon, hindbrain, spinal cord and pancreas, in a pattern reminiscent of
the pattern of pax6 expression in the mouse embryo
(MacDonald et al., 1994
;
Nornes et al., 1998
).
Interestingly, prominent expression of pax6.1 and pax6.2 was
not detected in the proliferating telencephalon of the zebrafish, in contrast
to mouse embryos (MacDonald et al.,
1994
; Stoykova et al.,
2000
; Wullimann and Rink,
2002
). The pax6-expressing cells in the zebrafish
telencephalon constitute a small population of migrating, post-mitotic cells
at the pallial/subpallial border. By contrast, pax6 is abundantly
expressed in the proliferating radial glia cells of the mouse telencephalon.
This suggests that the pattern of pax6 expression was modified during
vertebrate brain evolution.
Expression of ngn2 in the mouse depends on pax6 activity
in both the spinal cord and the telencephalon
(Stoykova et al., 2000;
Scardigli et al., 2003
). It
was recently shown that the ngn2 upstream region contains a
pax6-dependent regulatory region that drives expression in the spinal
cord (Scardigli et al., 2003
).
Moreover, ngn2 expression in the telencephalon of the mouse depends
on pax6 activity (Stoykova et
al., 2000
). Based on the expression of pax6.1 and
pax6.2 in the zebrafish neural plate/tube, pax6 may possess
equivalent functions in the control of the related ngn1 gene during
primary neurogenesis in the zebrafish embryo.
We have previously mapped the regulatory regions of ngn1
responsible for driving reporter expression in the neural plate. Two regions,
the lateral stripe element (LSE) and the anterior neural
plate element (ANPE) were identified in the ngn1
upstream region (Blader et al.,
2003). The LSE is required for expression in precursors
of Rohon Beard sensory neurones and reticulospinal neurones in the anlage of
the spinal cord and hindbrain, respectively; the ANPE is responsible
for expression in the ventral caudal cluster in the midbrain anlage, the
trigeminal ganglia and a few scattered nuclei in the anterior hindbrain
(Blader et al., 2003
). Further
analysis of ANPE showed that it contained an E-box known to interact
with bHLH factors. Indeed, Her5 was demonstrated to regulate the activity of
ANPE, as in embryos that lacked Her5, expression of a transgene that
contained the ANPE was expanded into the MHB area
(Geling et al., 2004
).
Moreover, mutation of the E-box in the ANPE caused an expansion of
reporter gene expression into the MHB area, suggesting that the E-box is
required for the suppression of transgene activity in the MHB by Her5
(Geling et al., 2004
).
These cis-regulatory regions show homology with sequences at the mouse and
human ngn1 loci, despite the fact that mammals do not express
ngn1 in the neural plate, but only later in the neural tube
(Blader et al., 2003;
Simmons et al., 2001
). This
suggests that these regions have shared functions in neurogenesis in mammals
and teleosts. The preliminary analysis of transgenes lacking the LSE
and ANPE in post-somitogenesis-stage zebrafish embryos suggested that
more proximal regions of the zebrafish ngn1 gene have regulatory
activity at later stages when the neural tube has formed
(Blader et al., 2003
).
To delineate the regulatory regions responsible for brain expression of
ngn1 in older zebrafish embryos, we analysed transgenic lines
carrying wild-type and deletion variants of ngn1 transgenes. We
mapped two regulatory regions that are required for transgene expression in
the brain of post-somitogenesis-stage embryos. The first region, residing at
position 6702 to 6490 bp upstream of the ATG start site, which
was also previously shown to harbour the LSE, drives expression in
the dorsal telencephalon. A second regulatory region referred to as
LATE was mapped to position 1775 to 1368. The
LATE region, like the LSE, is highly conserved in mouse and
human homologues of ngn1. We carried out comparative functional
studies in mouse embryos to investigate the activity of these conserved
regulatory elements. We focused on the dorsal telencephalon of the mouse, as
this is undoubtedly the most derived brain region to have arisen during
vertebrate evolution (Nieuwenhuys,
1994; Wullimann and Rink,
2002
). The LSE drives expression in the dorsal
telencephalon in both mouse and zebrafish embryos, indicating a conserved
function with respect to telencephalic expression. Curiously, we found that
the LATE region of the zebrafish ngn1 gene drives expression
in the lateral telencephalon of the mouse embryo but not in the zebrafish
telencephalon. The area of activity of LATE overlaps with that of the
paired-homeodomain transcription factor Pax6, suggesting a role of Pax6 in
regulating the activity of LATE. We demonstrate that Pax6 binds to a
conserved Pax6-binding site in the LATE region. Moreover, the lack of
pax6 activity in zebrafish by simultaneous knockdown of both
pax6.1 and pax6.2 leads to a small eye phenotype and
strongly reduces endogenous ngn1 and transgene expression. These
results are consistent with a direct regulatory role of Pax6 on the activity
of LATE. Based on the highly modular structure of vertebrate
regulatory regions, which are usually composed of multiple short and
degenerate binding sites for transcription factors, it is commonly assumed
that elaboration of novel patterns of gene expression is accomplished by
changes in the regulatory sequence
(Ludwig, 2002
;
Stone and Wray, 2001
;
Tautz, 2000
). Our data suggest
that a pre-existing enhancer was co-opted, and that the evolution of the
pax6 expression pattern led to the recruitment of LATE into
the newly developed territories of the mouse telencephalon.
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Materials and methods |
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Transgenic animals and morpholino knockdown
Reporter fragments for generating transgenic fish were excised from
plasmids and separated by agarose gel electrophoresis, followed by
purification with the Qiaex II Kit (Qiagen), according to the manufacturer's
instructions. Fragments were diluted to 50 ng/µl in TE and injected into
freshly fertilised zebrafish embryos, as previously described
(Blader et al., 2003). Mouse
transgenics were generated as previously described
(Scardigli et al., 2001
).
Morpholinos (GeneTools) were designed complementary to the 5' region of
pax6.1 (5'-TTTGTATCCTCGCYGAAGTTCTTCG-3') and
pax6.2 (5'-CTGAGCCCTTCCGAGCAAAACAGTG-3') mRNA. They were
resuspended in 1xDanieau buffer [58 mM NaCl, 0.7 mM KCl, 0.4 mM
MgSO4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES, pH 7.6]
and stored at 4°C. The morpholinos were injected into the yolk of
zebrafish embryos at the one- to two-cell stage, and at concentrations of
between 0.6 mM and 1.2 mM.
In situ analysis
In situ hybridisation and immunohistochemistry was performed using
ngn1 (Blader et al.,
1997) and gfp (Blader
et al., 2003
) antisense riboprobes and antibodies directed against
E.coli ß-galactosidase or GFP
(Scardigli et al., 2001
),
respectively.
Sequence comparisons
Human (AC005738), chicken (AY518341) and zebrafish (AF017301) sequences
were extracted from GenBank. Xenopus tropicalis homologous sequences
(scaffold_9761) were found by blasting the genome assembly one on the JGI
website
(http://genome.jgipsf.org/cgi-bin/runBlast?db=xenopus0).
Mouse sequences were kindly provided by J. Johnson
(Nakada et al., 2004).
Multiple alignment was performed using T-Coffee
(Notredame et al., 2000
). Pax6
putative-binding sites (Epstein et al.,
1994
) were searched using GCG findpattern (Accelrys).
Electrophoretic mobility shift assays
Pax6 protein was produced in vitro using the Sp6TNT kit (Promega) according
to the manufacturer's protocol. Oligonucleotides containing the site C
sequence (5'-GGCTTTGATATATCATACATGCCTGAAGACTCCC-3'), or clusters
of point mutations (shown in bold)
(5'-GGCTTTGATAGCGACGCACGTAAGTCCGACTCCC-3') were annealed by
heating to 90°C with an equimolar mixture of the upper and the lower
strands, and cooling slowly to room temperature. Annealed oligonucleotides
were labelled with [32ATP] using T4 polynucleotide kinase.
Binding reactions were performed in a total volume of 25 µl containing 10
mM Hepes (pH 7.9), 100 mM KCl, 4% Ficoll, 1 mM EDTA, 1 mM DTT and 2.5 µg of
poly(dI-dC). The reactions contained 4 µl of protein and 30,000 cpm of
probe. The reactions were allowed to proceed for 30 minutes at 4°C and
were analysed on a 6% polyacrylamide gel containing 0.25xTris
borate-EDTA (TBE).
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Results |
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Given that the complete pattern of endogenous Ngn1 expression can be obtained with the 8.4ngn1:gfp zebrafish transgene in mouse, regulatory sequences controlling lateral expression must be included in this sequence that are apparently inactive in the zebrafish telencephalon. Sequence comparison between the zebrafish and mammalian ngn1 loci revealed a region of homology located between 1775 to 1368 kb upstream of the zebrafish ngn1 start codon (called LATE; 61% homologous to mouse Ngn1; Fig. 7A). Consistent with the hypothesis of LATE being necessary for reporter expression in the lateral pallium of the mouse, E12.5 embryos transgenic for 3.1ngn1:nlacZ exhibited transgene activity in the telencephalon in a pattern very similar to that of 8.4(del3)ngn1:gfp transgenics (Fig. 3D,E). Deletion of the LATE region [3.1(delLATE)ngn1:nlacZ] rendered the transgene inactive in the telencephalon (Fig. 3F), indicating that LATE is indispensable for this activity in the mouse. Thus, expression of the 8.4ngn1:gfp zebrafish transgene in the telencephalon of mouse is a composite of the activities of two regulatory regions, LSE and LATE (Fig. 3G).
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To test whether LATE activity in the zebrafish embryo is also dependent on Pax6, as observed in the mouse, we knocked down Pax6 activity by injecting a cocktail of two antisense morpholinos complementary to pax6.1 and pax6.2 mRNA. Phenotypically, 60% (n=152) of the injected embryos showed a reduction in the size of their eyes (Fig. 6A,B), when compared with wild type. Expression of the endogenous ngn1 gene (Fig. 6D) (22%, n=41), as well as the 8.4ngn1:gfp transgene (28%, n=71, Fig. 6F), was significantly reduced in the diencephalon and hindbrain of morpholino-injected embryos. Expression in the telencephalon was not affected, indicating that the LSE is not dependent on Pax6 activity. The injection of either the pax6.1 or the pax6.2 morpholino alone did not cause an effect, suggesting that the two pax6 genes act redundantly. Moreover, the lack of an effect of each morpholino by itself, even at high concentrations (1.2 mM), is strongly suggestive of a specific interaction of the morpholinos with the pax6.1 and pax6.2 mRNA.
|
To test whether the three Pax6-binding site homologies in the LATE
region can bind Pax6 protein, we performed electromobility-shift assays with
recombinant mouse Pax6 protein that was synthesised by in vitro translation
(Scardigli et al., 2003).
Oligonucleotides comprising either site-A, site-B or site-C
(Fig. 7B) were
32P-labelled and incubated with recombinant Pax6 protein in the
presence of unlabelled oligonucleotide containing the homologous Pax6-binding
site, or an oligonucleotide in which the putative Pax6-binding site was
mutated by a cluster of point mutations. As a positive control, we used a
previously described consensus Pax6-binding site
(Czerny et al., 1993
). The
site-C oligonucleotide (sC) gave a strongly shifted band that was not reduced
by unspecific competitor with a mutated binding site (msC), but was totally
abolished by the presence of competitor oligonucleotide with an intact
Pax6-binding site homology (Fig.
8). Site-A and site-B did not yield retarded protein-DNA complexes
(data not shown), suggesting either that they do not interact with Pax6 or
that they bind to the protein very inefficiently.
|
In zebrafish embryos, the mouse LATE and LSE regions have the same regulatory activities as their cognate zebrafish enhancers
The zebrafish LATE region drives expression in the lateral
telencephalon of the mouse but not of the zebrafish. The high conservation of
LATE suggests that it may be the target of similar regulatory
principles that are used in different places in the forebrain of the zebrafish
and the mouse. Its dependence on Pax6 activity in both mouse and zebrafish is
in support of this notion. Hence, one prediction is that the mouse
LATE region, like the zebrafish LATE region should be active
in the zebrafish diencephalons, but should not drive expression in the
telencephalon.
To test this hypothesis, zebrafish embryos were injected with constructs,
in which the zebrafish LATE was replaced with the mouse element
(Fig. 9). The embryos were
analysed at 26 hours after injection. To overcome the moscaism of such
transient expression patterns, we used the SceI meganuclease protocol
(Thermes et al., 2002), and
collected, in addition, accumulative expression maps by overlaying the
expression pattern from many independently injected embryos
(Fig. 9A-F). The replacement of
zebrafish LATE with the conserved mouse LATE sequences
produced embryos in which the expression of GFP was restricted to the
diencephalon (Fig.
9C,C'), in a pattern similar to that of the zebrafish
LATE region (Fig.
9A,A'). This is reminiscent of the spatial activity of the
3.1ngn:gfp stable transgenic lines
(Fig. 4A). In addition,
injected embryos do not show expression in the telencephalon
(Fig. 9C,C'). As seen in
the stable transgenic lines, deletion of LATE abolished diencephalic
expression almost completely (Fig.
9B,B'). Taken together, this suggests that mouse
LATE is functionally similar to zebrafish LATE when
introduced into the zebrafish embryo. Moreover, these findings suggest that
the regulatory principles controlling LATE in the zebrafish
diencephalon were co-opted in the evolution of the lateral telencephalon of
the mouse. There is, however, some variation in the diencephalic pattern
driven by the mouse and zebrafish LATE enhancers, indicating that
changes have occurred during evolution at the level of fine-tuning of the
expression pattern within the diencephalon.
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Discussion |
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Pax6 as a pre-pattern gene in the zebrafish brain
Several lines of evidence suggest that Pax6 is a regulator of
LATE. First, the pattern of expression of pax6 precedes and
subsequently overlaps with that of LATE activity in the dorsal
diencephalon and in the hindbrain. Moreover, LATE contains a
pax6-binding site that is conserved in mammalian homologues of
ngn1 and that binds recombinant Pax6 in vitro. The simultaneous
knockdown of pax6.1 and pax6.2 in the zebrafish embryo leads
to the reduction of ngn1 and transgene expression in the hindbrain
and diencephalon, in a manner very similar to that observed following the
deletion of LATE from transgenes. Finally, transgene expression in
the mouse depends on a functional pax6 gene.
Like the pre-pattern genes her5, iroquois1 and iroquois7
(Geling et al., 2003;
Itoh et al., 2002
), the
zebrafish pax6 genes (Krauss et
al., 1991
; Nornes et al.,
1998
) are expressed in broad domains in the neural plate from
early stages onwards. However, not all cells within these broad domains
express the transgene or the endogenous ngn1 gene. Rather
ngn1 expression is restricted to distinct clusters of neurones that
are located in at least six regions of the diencephalon, including
non-pax6-expressing territories. In particular, the transcripts are
detected in the epiphysis, pretectum, dorsal thalamus, ventral thalamus,
preoptic area and the posterior tuberculum, in contrast to the expression of
pax6.1 and pax6.2, which is predominantly located in the
alar plate of the forebrain. This suggests that pax6 genes define a
broad domain of competence in the diencephalon, in which other factors
cooperate to specify the precise sub-region of neurogenesis. Hence,
pax6 could be regarded to act as a pre-pattern gene in the zebrafish
neural plate/tube, in a similar fashion to iroquois or her5
genes.
Humans and mice carrying a loss-of-function allele of Pax6 show a
severe haplo-insufficiency, causing aniridia and small eye phenotypes,
respectively (Engelkamp and van Heyningen,
1996). This seems to be in contrast to the situation in zebrafish
embryos, where reduction of pax6 activity by knockdown of an
individual pax6 gene did not cause a visible effect in the embryo.
This suggests that the two pax6 genes in the zebrafish can act
redundantly, at least during early embryonic stages. However, knockdown of
gene function by a morpholino approach can impair gene function only
transiently: defects obvious at later stages could thus not be scored.
Expression of the mouse Ngn2 gene is dependent on Pax6
activity in the telencephalon and in the spinal cord
(Stoykova et al., 2000;
Scardigli et al., 2003
). Mouse
Ngn1 and Ngn2 are expressed in similar but not identical
patterns in the mouse nervous system, suggesting that the two Ngn
genes are regulated by related mechanisms in the mouse that are derived from a
common ancestral gene. However, despite the strong conservation of other
regulatory sequences, such as the ANPE
(Blader et al., 2003
), the
zebrafish LATE enhancer sequence is not conserved in
Ngn2.
The recruitment of LATE in the mouse appears to be linked to evolution of the telencephalon
The telencephalon is one region of the brain where molecular changes in
regulatory activity are likely to have occurred most extensively, given the
vast expansion and morphological variations of these forebrain structures
among different vertebrate groups
(Nieuwenhuys, 1994). These
differences are particularly striking in regions flanking the pallial (dorsal
telencephalon)-subpallial (ventral telencephalon) boundary, which plays a
pivotal role in the establishment of neuronal diversity, and in the reception
of diverse developmental signals from dorsal and ventral domains of the
telencephalon (Molnar and Butler,
2002
). Despite the anatomical differences in telencephalon
structure, comparative gene expression studies suggest that development of the
forebrain follows a similar `Bauplan' in all vertebrates, raising the question
of how morphological differences evolved
(Fernandez et al., 1998
;
Reiner, 2000
;
Striedter, 1997
;
Zerucha et al., 2000
).
ngn1 is expressed in the dorsal telencephalon in both zebrafish and
mouse (Blader et al., 2003
;
Fode et al., 2000
), and thus
represents an interesting case to study the modification of cis-regulatory
elements during evolution of the telencephalon.
We demonstrate that expression of ngn1 transgenes in the dorsal
telencephalon of zebrafish embryos is dependent on the activity of one
regulatory region (LSE), whereas expression in the developing
isocortex of mouse requires the activity of two distinct regulatory regions
(LSE and LATE). The zebrafish regulatory sequences
faithfully recapitulate the endogenous pattern of Ngn1 expression in
the mouse, including the position of the sharp ventral boundary and the
high-lateral to low-medial gradient in the pallium. LATE is highly
conserved in the murine Ngn1 and Ngn2 genes
(Blader et al., 2003;
Scardigli et al., 2003
). The
homologous region of Ngn2 is a direct target of Pax6 and
also drives expression in the lateral telencephalon
(Blader et al., 2003
;
Scardigli et al., 2003
). As
LATE is not required for lateral telencephalon expression in the
zebrafish, what maintained LATE over 450 million years of evolution?
Our data show that zebrafish LATE is employed to drive expression in
the diencephalon of the zebrafish, and that this function has also been
retained by the mouse LATE when placed into the context of the
zebrafish embryo.
Evolutionary modification of expression patterns
Eukaryotic regulatory regions are usually composed of multiple
protein-binding sites clustered within a few hundred base pairs or less. The
internal organisation of these regions can be rather flexible, as individual
protein-binding modules can vary in position and orientation, and the actual
DNA sequences bound by regulatory proteins are usually rather short and
degenerate. In addition, multiple regulatory regions that can be scattered
over megabases in vertebrate genomes contribute frequently to the expression
of a gene (Davidson et al.,
2000). Given this flexibility in the cis-regulatory organisation
of vertebrate genes, the strong conservation of the position and sequences of
the individual regulatory regions (LSE, ANPE, LATE) of the
ngn1 gene is remarkable. Regulatory regions of different genes of the
same species can change during evolution at varying speeds
(Davidson, 2001
). The
conservation of regulatory regions over 450 million years of independent
evolution is not only restricted to ngn genes but also includes other
regulators of neurogenesis, such as delta-d
(Dornseifer et al., 1997
),
sonic hedgehog (Müller et
al., 2002
; Müller et al.,
1999
) and pax6
(Kammandel et al., 1999
),
which is suggestive of a strong selection pressure to maintain the structure
of the regulatory regions of some genes.
It is believed that the evolutionary diversification of the body plan was
driven to a large extent by changes in gene expression, rather than by the
emergence of novel regulatory proteins
(Dermitzakis and Clark, 2002;
Ludwig, 2002
;
Stone and Wray, 2001
;
Tautz, 2000
). In principal,
three ways of how the cis-regulatory region evolved can be envisaged.
Cis-regulatory regions could have emerged de novo by the accidental clustering
of protein-binding modules in close proximity in non-coding sequences.
Alternatively, existing regulatory regions could have been modified by the
deletion or addition of protein-binding sites, giving novel patterns of
regulatory activity. As a third possibility, enhancer sequences could have
been co-opted. In this case, the expression of the interacting transcription
factors was altered, placing a pre-existing enhancer into a different spatial
and/or temporal regulatory context. In addition, rearrangements of the genome
may have played crucial roles in redistributing these novel regulatory
activities among genes.
Our findings for the ngn1 enhancers suggest a scenario in which a
regulatory sequence has been co-opted in order to drive expression in a novel
context. Pax6 is not widely expressed in the telencephalon of early
post-somitogenesis-stage zebrafish embryos. Only a few migratory post-mitotic
telencephalic cells express Pax6 in the zebrafish embryo
(Wullimann and Rink, 2002).
This is in striking contrast to the widespread expression of Pax6 in the
proliferative radial glia in the telencephalon of the mouse embryo
(Gotz et al., 1998
;
Stoykova et al., 2000
). This,
together with the dependence of LATE activity on Pax6, indicates that
evolutionary modulation of Pax6 expression could have been involved in the
recruitment of LATE for Ngn1 expression in the lateral
telencephalon of the mouse. Co-option of a regulatory sequence to drive
expression at the pallial/subpallial border and in immediately adjacent
structures in the mouse is particularly intriguing, as this region is believed
to be absent from the zebrafish telencephalon, and to have appeared first in
the amniote lineage as a major prerequisite for the emergence of the cortex in
mammals (Molnar and Butler,
2002
). However, it is unlikely that Pax6 is the only factor
involved in this recruitment. Other regions of the embryo that express Pax6 do
not show expression of endogenous ngn1 or the
LATE-containing transgenes. Moreover, even in the small group of
cells that express pax6 in the zebrafish telencephalon, Pax6 is not
sufficient to activate ngn1 expression, indicating that cooperating
factors are also necessary. This is reflected in the extended regions of
conserved sequence flanking the Pax6-binding site in LATE that
presumably represent the phylogenetic footprints of other conserved
transcription factors (see Fig.
7). Nevertheless, our data, together with the appearance of
extensive Pax6 expression in the radial glial cells of the mammalian
neocortex, suggest an important role of Pax6 as one of the factors that have
recruited Ngn1 expression in the telencephalon of mammals.
Conclusion
Our results provide evidence that the co-option of pre-existing enhancers
is a mechanism to diversify regulatory patterns during evolution. These
results have further implications: the attempts to delineate fields of
evolutionary homology on the basis of shared gene expression can be
misleading, as expression territories may be composites of the activities of
distinct regulatory regions that have evolved independently, as demonstrated
here for the LSE and the LATE regions of ngn1.
Moreover, in comparative genomic approaches to identify regulatory regions by
sequence conservation, the regulatory function cannot be inferred from
conserved sequences, as regulatory regions may have been co-opted in distinct
processes during evolution.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Centre de Biologie du Developpement, bat 4R3, Universite
Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France
Present address: Institute of Cell Biology and Tissue Engineering, Via di
Castel Romano 100/102, 00128 Rome, Italy
Present address: Division of Molecular Neurobiology, National Institute for
Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
¶ Present address: 2277 HSC, University of Calgary, 3330 Hospital Drive NW,
Calgary, Alberta T2N 1N8, Canada
** Present address: Institute for Toxicology and Genetics, Forschungszentrum
Karlsruhe, Postfach 3640, 76021 Karlsruhe, Germany
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