1 Department of Cell Biology, Biozentrum, University of Basel,
Klingelbergstrasse 70, CH-4056 Basel, Switzerland
2 Institut de Génétique Humaine, Centre National de la Recherche
Scientifique UPR 1142, 141 rue de la Cardonille, 34396 Montpellier,
France
3 Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka,
Suita, Osaka 565-0871, Japan
* Author for correspondence (e-mail: Walter.Gehring{at}unibas.ch)
Accepted 3 February 2005
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SUMMARY |
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Key words: Crystallin, Enhancer conservation, Pax6, Pax2, Sox2, SoxN, Drosophila
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Introduction |
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Lens development has long been used as a model system for the study of
tissue differentiation. Complex eyes with lenses exist in a wide range of
animals, from vertebrates to invertebrates
(Tomarev and Piatigorsky,
1996; Piatigorsky,
2003
). In all cases, the lenses are transparent structures, the
primary function of which is to refract light on to the retina. Lens
differentiation is accompanied by the expression of several lens-specific
genes, such as Crystallins, which encode structural proteins responsible for
the transparent and refractive properties of the lens. The Crystallin proteins
accumulate in the lenses and can account for 80-90% of the water-soluble
protein content of the lens (Piatigorsky,
2003
). Although all Crystallin proteins fulfil a similar function,
comparative analysis has revealed an unexpected heterogeneity and diversity
among the members of this family
(Piatigorsky, 1993
). The
vertebrate Crystallins can be divided into two groups: ubiquitous Crystallins
and taxon-specific Crystallins. The former are present in all major vertebrate
lenses and show sequence similarity to stress proteins. The latter are
restricted to certain taxonomic groups or species, and are related or
identical to metabolic enzymes (Wistow and
Piatigorsky, 1998
;
Piatigorsky, 2003
). The
invertebrate Crystallins have not been studied so extensively. Nevertheless,
some of them have been molecularly characterized and also show sequence
similarity to metabolic enzymes
(Piatigorsky, 2003
). In
Drosophila, one of the Crystallin proteins from the corneal lens was
isolated and showed to be related to insect cuticle proteins
(Komori et al., 1992
;
Janssens and Gehring,
1999
).
Despite Crystallin heterogeneity, many studies have shown that most
Crystallin genes are regulated by a small set of evolutionarily conserved
transcription factors (Cvekl and
Piatigorsky, 1996). The chicken
1-crystallin is
one of the best-characterized Crystallin genes. It is a taxon-specific
Crystallin present in birds and reptiles
(Wistow and Piatigorsky,
1987
). Its lens-specific regulation is under the control of the
DC5 fragment located within the 1 kb-long intronic enhancer. The DC5 fragment
is just 30 bp long and contains both a PAX6 and a SOX2 binding site. Extensive
in-vitro and in-vivo analyses have demonstrated that DC5 activity depends on
the synergistic action and cooperative binding of PAX6 and SOX2 to the DC5
fragment (Kamachi et al.,
2001
). PAX6 is a member of the Pax protein family and contains two
DNA-binding domains: a paired domain and a homeodomain. It is considered a
master regulator of eye development
(Gehring and Ikeo, 1999
) and a
key transcription factor in vertebrate lens development
(Cvekl and Piatigorsky, 1996
).
In addition, it is also necessary for the development of the nose and pancreas
and parts of the central nervous system. SOX2 is a member of the Group B1
subfamily of Sox transcription factors. SOX proteins bind to DNA in a
sequence-specific manner by means of a high-mobility group (HMG) domain, and
are involved in a variety of developmental processes, either activating or
repressing specific target genes through interaction with different partner
proteins (Kamachi et al.,
2000
; Wilson and Koopman,
2002
). It has been proposed that partnering with co-DNA-binding
factors is the mechanism SOX proteins use to distinguish their regulatory
targets and act in a cell-type-specific fashion
(Kamachi et al., 2000
).
To test the idea that Pax6 and Sox2, together with the
DC5 enhancer, could form a basic regulatory circuit functional in distantly
related animals, we introduced the DC5 enhancer into Drosophila and
studied its activation pattern and regulation in the eye field. The
Drosophila compound eye is made up of approximately 800 identical
units called ommatidia. Each ommatidium contains a set of retinal cells,
consisting of eight photoreceptors, 12 accessory cells and a lens. The lens
has two parts: the corneal lens and the crystalline cone, and it accumulates
Crystallin proteins secreted by the underlying cone cells and primary pigment
cells (Wolff and Ready,
1993).
The results presented in this report show that the DC5 enhancer is not only
active in the Drosophila compound eye but, remarkably, is
specifically active in those cells responsible for Crystallin secretion, i.e.
the cone cells. However, regulation of the DC5 enhancer is carried out not by
Pax6, but by Pax2 (D-Pax2; shaven
FlyBase) in combination with the Sox2 homologue SoxNeuro
(SoxN). PAX2 and PAX6 are closely related Pax proteins. Both proteins
recognize almost identical consensus sequences with their paired domain
(Czerny and Busslinger, 1995),
and to some extent are functionally interchangeable in Drosophila
(Kozmik et al., 2003
).
These results suggest that, despite evident anatomical, developmental and physiological differences between the vertebrate eye and the Drosophila compound eye, basic genetic regulatory circuits involved in the development of these two evolutionarily diverged eyes have been largely conserved.
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Materials and methods |
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SoxN loss-of-function clones were generated by Flp-mediated
mitotic recombination using the null allele SoxNU6-35 and
the FLP/FRT system (Xu and Rubin,
1993). Flies with the genotype w;
SoxNU6-35 FRT40A/CyO were crossed to w
Bac([P3-DsR]-DC5(8x)wt); P(w+mC)36F
FRT40A/P(w+mC)36F FRT40A; ey-flp/ey-flp
and the offspring analysed to detect mutant clones in the compound eye.
DNA constructs and transgenic flies
Wild-type (wt) and mutant forms (M4 and M7) of the octamerized DC5 enhancer
were cloned as EcoRI fragments into the vector pSLfaGFPfa, upstream
of a Drosophila basal promotor (hsp27 heat-shock promoter)
and the EGFP reporter gene. The vector pSLfaGFPfa was constructed by cloning
the fragment EcoRI-hsp27-EGFP-SV40polyA-HindIII
into the plasmid pSLfa1180fa (Horn and
Wimmer, 2000). The `cassettes' containing
DC5(8x)-hsp27-EGFP-SV40polyA were then isolated as AscI
fragments and cloned into the piggyBac transposon derived vector
pBac[3xP3-DsRedaf]. This vector contains the DsRed1 gene, under the
control of the artificial 3xP3 eye promoter, as a transgenesis marker
(Horn et al., 2002
). The
resulting plasmids pBac[P3-DsR]-DC5(8x)wt, pBac[P3-DsR]-DC5(8x)M4 and
pBac[P3-DsR]-DC5(8x)M7 were used to generate Drosophila transgenic
lines by germline transformation in yw1118. The EGFP
modified version, containing the Drosocrystallin signal peptide in its
N-terminus (SP+EGFP), was obtained by standard PCR techniques. The wild-type
EGFP was then substituted by its modified version within the plasmid
pBac[P3-DsR]-DC5(8x)wt, and the resulting plasmid
pBac[P3-DsR]-DC5(8x)wt-SP+EGFP used for germline transformation in
Drosophila. The SME enhancer (a minimal D-Pax2 eye-specific
enhancer) (Flores et al.,
2000
) was amplified by PCR and cloned as an EcoRI
fragment into the vector pSLfaRFPfa. This vector is similar to pSLfaGFPfa but
contains mRFP (Campbell et al.,
2002
) instead of EGFP. The `cassette'
SME-hsp27-mRFP-SV40polyA was isolated as an EcoRI (partial
digestion)-AscI fragment and used to substitute the 3xP3-basal
promoter-EGFP-SV40polyA `cassette' present in the Hermes transposon
derived vector pHer[3xP3-EGFPaf] (Horn et
al., 2000
), giving rise to the plasmid pHer[SME-mRFPaf]. Then, the
`cassette' DC5(8x)wt-hsp27-EGFP-SV40polyA was cloned as an
AscI fragment into the vector pHer[SME-mRFPaf], and the resulting
plasmid pHer[SME-mRFP]-DC5(8x)wt used for germline transformation. The
transgenic flies harbouring this construct express mRFP under the control of
the SME enhancer and EGFP under the control of the DC5(8x) enhancer. A DNA
fragment containing the promoter and the 5' untranslated region of
SoxN (from 2939 to +869. PSoxN) was
amplified by PCR and cloned as an EcoRI-NcoI fragment into
the vector pSLfaGFPfa. The use of the NcoI site eliminates the
hsp27 basal promoter present in pSLfaGFPfa. Then the `cassette'
containing PSoxN-EGFP-SV40polyA was isolated as an
AscI fragment and cloned into the vector pHer[SME-mRFPaf]. The
resulting plasmid pHer[SME-mRFP]-PSoxN was used for
germline transformation in Drosophila. The transgenic flies
containing this construct express mRFP under the control of the SME enhancer
and EGFP under the control of the PSoxN promoter. Detailed
descriptions of the primers used for the cloning procedure described above are
available upon request.
The UAS-SoxN construct was made as follows: a genomic P1 clone containing the complete SoxN gene was digested with NheI, filled in with Klenow and digested with NotI, and introduced into pCasper cut with EcoRI/klenow and NotI. The resulting construct contains the complete SoxN open reading frame flanked by 0.75 kbp 5'UTR.
For the UAS-DsRed1 construct, a BamHI-XhoI fragment containing the DsRed1 gene was isolated and cloned into pUAST cut with BglII and XhoI.
Cryosections
Young adult flies (within 1 day after hatching), expressing fluorescence
proteins in the compound eyes, were beheaded under anaesthesia. Heads were
then imbedded in OCT compound (Miles) and frozen in liquid N2.
Sections of 10 µm were cut with a cryostat, dried at 50°C for 2 minutes
and directly analysed with a fluorescence microscope.
Protein expression and DNA binding assays
The following recombinant proteins were produced and purified from
Escherichia coli according to manufacturer's instructions. The amino
acids involved in each construct are shown in brackets: EY-PD (37-166), TOY-PD
(29-156) and D-PAX2-PD (175-302) were tagged with 6xHis at its N-terminus
(Qiagen). D (100-382) and SOXN (158-261) were expressed as N-terminal GST
fusion proteins (Amersham Pharmacia Biotech).
Gel mobility shift assays were used to study protein-DNA interactions.
Probes containing wild-type or mutant DC5 sequences, binding reactions and gel
electrophoresis conditions are described in Kamachi et al.
(Kamachi et al., 2001).
Cell culture and transfection assays
Reporter vectors were constructed by cloning the wild-type and mutant
DC5(8x) enhancers in the plasmid pLacZH. pLacZH is a modified version of
pLacZi (Clontech), in which the yeast minimal promoter
PCYC1 was replaced by the Drosophila minimal
promoter hsp27. The expression vectors were constructed by cloning
the cDNAs of the tested genes (ey, toy, D-Pax2, D and SoxN)
into the vector pAc5.1B/V5His (Invitrogen) under the control of the
constitutively active actin5c promoter. For reporter gene assays
1.5x106 Drosophila Schneider 2 cells were
transfected with a total of 200 ng of DNA (20 ng reporter plasmid, 5 ng of a
plasmid constitutively expressing firefly luciferase, 50 ng of expression
plasmids and pAc5.1B/V5His to bring total DNA to 200 ng) using the Effectene
Transfection Reagent (Qiagen).
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Results |
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DC5 is active in the cone cells of the adult Drosophila compound eye and its precursors in the larval eye imaginal disc
The DC5 fragment contains elements sufficient to elicit lens-specific
enhancer action in its multimeric form in chicken cells
(Kamachi and Kondoh, 1993).
Accordingly, an octamerized version of the DC5 enhancer was introduced into
Drosophila upstream of the reporter gene EGFP and a minimal promoter
(Fig. 1B). DsRed1 under the
control of the synthetic promoter 3xP3
(Horn et al., 2000
) was used
as a marker for transgenesis. Two mutant forms of the enhancer representing
the most stringent SOX2 site and PAX6 site mutations (M4 and M7, respectively)
(Fig. 1A)
(Kamachi and Kondoh, 1993
)
were also octamerized and introduced in the same way. In these transgenic
flies, the wild-type DC5 enhancer was active in the adult compound eye, but
not in the ocelli (Fig. 1C).
Furthermore, the two mutant forms of DC5 showed no enhancer activity
(Fig. 1D,E), suggesting that
the integrity of both binding sites is also important for the enhancer action
in Drosophila. Additional EGFP expression was also detected in the
antennae of young flies and in the adult mouthparts (the labial and maxillary
palps) (Fig. 1I). The activity
of the DC5 enhancer was traced back during Drosophila development.
Enhancer action was first detected in the eye imaginal disc during the third
instar larva, in cells posterior to the morphogenetic furrow (MF)
(Fig. 1J). Expression was also
detected in the larval visual system (Bolwig's organ)
(Fig. 1K,L). In the adult
compound eye, DC5 activity was found in non-neuronal cells under the lenses,
in a position resembling the one occupied by the cone cells. In order to
determine the exact nature of these cells, we compared the EGFP signal with
the pattern of mRFP expressed under the control of the SME enhancer. SME is
specifically active in cone cells and corresponds to the minimal eye-specific
enhancer of the Drosophila Pax2 homologue D-Pax2
(Flores et al., 2000
). The
results (Fig. 2A-J) showed a
clear co-localization of both fluorescent proteins, which unambiguously
identified the cells in which DC5 is active as cone cells. The fact that EGFP
signal was detected neither in the corneal lens nor in the crystalline cone
illustrates how these lens structures are formed in Drosophila. By
contrast to vertebrates, where lens formation is a cellular process, in
Drosophila the lenses are secreted by the cone cells and the primary
pigment cells into an acellular space on the top of the ommatidia. To verify
this, we modified the EGFP protein, inserting a signal peptide in its
N-terminus, and analysed the location of the fluorescent protein in transgenic
flies expressing the signal-peptide-tagged EGFP under the control of the DC5
enhancer. The results showed that the EGFP signal localized in the compound
eye lenses, corroborating the identity of the cells responsive to the DC5
enhancer as lens secreting cells (Fig.
2K,L).
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In summary, we can conclude that in vitro Drosophila PAX6/2 and SOX2 homologues can bind cooperatively to the DC5 enhancer.
Drosophila Pax6/2homologues activate DC5 in vivo when Sox2 homologues are co-expressed
We then examined whether the cooperative binding to DC5 detected among the
studied proteins resulted in a synergistic activation of the enhancer ex vivo.
To test this possibility, we carried out cell culture co-transfection assays
using Drosophila Schneider 2 (S2) cells. A reporter plasmid
containing the DC5 enhancer upstream of the ß-galactosidase gene
was co-transfected with effector vectors expressing Drosophila Pax6,
Pax2 and Sox2 homologues
(Fig. 4A1). When these genes
were separately expressed, the enhancer was only very modestly activated
(Fig. 4A2). High-level
activation was detected only when combinations of Pax6/2 and
Sox2 homologues were co-transfected at the same time, and activation
of the DC5 enhancer reached the highest level when ey, toy or
D-Pax2 were co-expressed with SoxN
(Fig. 4A2). When the mutant
forms of the enhancer were used, the activation levels remained almost basal,
even when combinations of Pax6/2 and Sox2 homologues were
co-expressed (Fig. 4A3,A4).
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DC5 activation in the Drosophila compound eye is attained by synergism of D-Pax2 and SoxN
The data presented above indicate that several combinations of
transcription factors can be responsible for activating the DC5 enhancer in
vivo in the Drosophila compound eye, e.g. ey/SoxN, toy/SoxN,
D-Pax2/D and D-Pax2/SoxN
(Fig. 4B4,B5,B6,B7). However, a
major constraint to their effective involvement in this process is their
expression pattern. The expression of Drosophila Pax6 homologues in
the adult compound eye is controversial and has not been firmly demonstrated.
Furthermore, their expression in the third instar eye imaginal disc during
larval development is restricted to undifferentiated cells anterior to the MF,
whereas the DC5 enhancer is active posterior to the MF in cone cell precursors
(Fig. 2). By contrast,
D-Pax2 expression in cone cell precursors of the eye imaginal disc
during the 3rd instar larval stage has been well documented
(Fu and Noll, 1997). As
revealed by the activity of its minimal eye-specific enhancer SME,
D-Pax2 is expressed not only in those cone cells precursors
(Fig. 2F), but also in the cone
cells of the adult compound eye (Fig.
2C,I). This expression pattern overlaps with the activity profile
displayed by the DC5 enhancer (Fig.
2E,H).
D and SoxN expression in the eye imaginal disc has also
been described. D is expressed anterior to the MF, along the
ventro-lateral region of the eye-antennal disc
(Mukherjee et al., 2000), in a
domain where the DC5 enhancer is not active. However, immunostaining reveals
expression of SoxN in cells posterior to the MF
(Crémazy et al., 2001
),
in the same domain in which the DC5 enhancer is active. Expression of
D and SoxN in the adult compound eye has not been described.
However, we have found that a 3.4 kb DNA fragment containing the promoter and
the 5' untranslated region of SoxN (PSoxN)
(Fig. 5A) harbours regulatory
sequences that recapitulate SoxN expression in the eye imaginal disc
during larval development (Fig.
5C). These sequences drive expression of a reporter gene in cells
posterior to the MF, which, by comparison to the SME enhancer activity
pattern, were identified as cone cells
(Fig. 5D,G,H). The
PSoxN fragment also drove expression of a reporter gene in
the adult compound eye (Fig.
5B).
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Discussion |
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The simplicity of the DC5 fragment, the well-characterized nature of its
transcription factor binding sites, and the fact that Pax6 and
Sox2 are important developmental regulators conserved in evolution,
prompted us to consider these three elements (DC5 sequence, Pax6 and
Sox2) as part of a conserved regulatory circuit involved in lens
development. To test this idea, we performed a functional enhancer test and
introduced the DC5 fragment into a distantly related organism, D.
melanogaster. Functional conservation of enhancer elements has been
previously reported. Exchanges of Hox and Pax6/eyeless enhancer
elements between flies, worms and vertebrates gave rise to expression patterns
that were characterized as homologous
(Streit et al., 2002;
Frasch et al., 1995
;
Xu et al., 1999
). In other
cases, enhancer elements from a variety of D. melanogaster neuronal
and muscular genes failed to activate the expression of a reporter gene in the
homologous cell types in Caenorhabditis elegans
(Ruvinsky and Ruvkun, 2003
).
These various outcomes are probably due to differences in the evolutionary
pressure exerted on different enhancers according to their developmental
roles. However, these results emphasize the importance of this test when the
result is positive, meaning that the functional conservation of an enhancer is
a reliable way to identify basic regulatory circuits.
The fruit fly and the chicken are separated by hundreds of millions of years of evolution, and their visual organs reflect this evolutionary distance at the anatomical, developmental and physiological level. Even the eye lenses, although fulfilling a similar function, are formed differently. In chicken the lenses are cellular structures, whereas in Drosophila they are secreted into an acellular space by the cone cells and the primary pigment cells. The introduction of the chicken DC5 enhancer into Drosophila had a remarkable effect. Not only was the DC5 enhancer active in the Drosophila compound eye, but also it was specifically active in the cells that are in part responsible for lens secretion in Drosophila, i.e. the cone cells. The experiment was done with an octamerized version of the DC5 enhancer to augment the sensitivity of the system. Actually, when a single copy of the DC5 enhancer was used, no activity was detected in the Drosophila compound eye (data not shown). This suggests that although lens-specificity is retained by DC5 in Drosophila, additional sequences have to be present to provide full activity to the enhancer.
DC5 regulation is under the control of D-Pax2 and SoxN in Drosophila
Once demonstrated that the DC5 enhancer is active in Drosophila,
we focused our attention on finding out whether the other two elements of the
regulatory circuit were also conserved, i.e. whether DC5 activity in
Drosophila was due to the synergistic action of Pax6 and
Sox2. Gel mobility shift assays showed that Drosophila PAX6
homologues (EY and TOY) and SOX2 homologues (D and SOXN) could bind
cooperatively to the DC5 sequence. Interestingly, the PAX6 paired domain was
sufficient for DNA binding and cooperation with D and SOXN. Cell culture
co-transfection assays and ectopic activation of the DC5 enhancer corroborated
these findings, and showed that the DC5 enhancer was synergistically activated
upon co-expression of Drosophila Pax6 (ey or toy)
and SoxN. However, an important constraint to the real involvement of
these transcription factors in the regulation of the DC5 enhancer in vivo is
their expression pattern. Whereas SoxN expression was detected in
both the adult compound eye and in cone cell precursors of the eye imaginal
disc (Fig. 5B,C), expression of
Drosophila Pax6 homologues in the adult compound eye is controversial
and has not been clearly demonstrated. In the eye imaginal disc, during the
third larval stage, Drosophila Pax6 homologues are expressed in
undifferentiated cells anterior to the MF, whereas DC5 is active posterior to
the MF in cone cell precursors. However, Drosophila Pax2, a gene
evolutionarily related to Pax6, is expressed in cone cells, primary
pigment cells and bristle cells of larval and pupal eye discs
(Fu and Noll, 1997). In
addition, a D-Pax2 cone-cell-specific enhancer has been characterized
in the fourth intron of the gene (Fu et
al., 1998
; Flores et al.,
2000
). This enhancer (called SME) is active in cone cells of the
adult compound eye and their precursors in the eye imaginal disc
(Fig. 2F,I), mimicking the
activity pattern of DC5. In-vitro and in-vivo studies showed that D-PAX2 could
cooperate with SOXN in binding to the DC5 sequence (indeed it showed a higher
affinity for DC5 than Drosophila PAX6 homologues), and thus activate
the enhancer in a synergistic fashion. Finally, loss-of-function analysis
showed conclusively that depletion of either D-Pax2 or SoxN
abolished DC5 activity in vivo, clearly demonstrating the involvement of these
two transcription factors in the activation of the DC5 enhancer in the
Drosophila compound eye.
It is interesting to point out that, apart from the compound eye, the DC5
enhancer is also active in other tissues of the adult fly, such as the antenna
and the mouthparts (the labial and maxillary palps). It is not known whether
Drosophila Pax6, Pax2 and Sox2 homologues are expressed in
these tissues of the adult fly, although D-Pax2, D and SoxN
expression has been detected in the developing antennal disc
(Fu and Noll, 1997;
Mukherjee et al., 2000
;
Crémazy et al., 2001
),
and ey has been shown to be involved in the development of the fly
maxillary palps and antennae (Benassayag et
al., 2003
). This opens the possibility of different combinations
of transcription factors taking part in the regulation of the DC5 enhancer in
other tissues besides the eye, and suggests that partnering among these
factors might also be used for developmental processes other than eye
development. In addition, the fact that several of these combinations can
effectively activate the DC5 enhancer in an ectopic situation
(Fig. 4B4,B5,B6,B7) further
supports this assumption. Interestingly, we could detect that the
cone-cell-specific enhancer SME also drove expression of a reporter gene in
the non-compound-eye tissues in which DC5 is active, namely the adult antenna
and the labial and maxillary palps (data not shown). Therefore, we favour the
hypothesis that D-Pax2 is also responsible for the activity of the
DC5 enhancer in these fly appendages.
Pax6 function in vertebrate lens development was taken over by Pax2 in Drosophila
The results obtained with the analysis of the chicken DC5 enhancer in
Drosophila have important evolutionary implications. They suggest
that Pax6 function in vertebrate lens development was probably
retained by Pax2 in Drosophila. PAX2 and PAX6 are closely
related proteins. They recognize almost identical consensus sequences with
their paired domains (Czerny and
Busslinger, 1995) and to some extent can be considered as
functional homologues in Drosophila, as D-Pax2 is able to
induce ectopic eyes (Pax6 function) and ey and toy
can rescue spapol mutation (Pax2 function)
(Kozmik et al., 2003
). In
agreement with this, a recently characterized cnidarian PaxB gene was
tentatively identified as the descendant of the last common ancestor of the
Pax6 and Pax2 genes
(Kozmik et al., 2003
;
Gehring, 2004
;
Piatigorsky and Kozmik, 2004
).
Like PAX2, PAXB protein contains a PAX2/5/8-type paired domain and
octapeptide; and, like PAX6, a complete paired-type homeodomain. As a
consequence, PaxB is able to rescue the Drosophila
spapol mutation (Pax2 function) and to induce small
ectopic eyes in Drosophila (Pax6 function). Interestingly,
PaxB is also able to activate the jellyfish J3-crystallin
promoter in cell culture co-transfection assays
(Kozmik et al., 2003
). It is
tempting to speculate that after duplication and diversification of the
ancestor PaxB-like gene, Crystallin regulation was retained by
Pax6 in the vertebrate lineage, whereas this function was taken over
by Pax2 in Drosophila. We can speculate further and suggest
that this functional diversification was probably due to changes in the
regulatory elements of these two genes and not in their coding sequences. At
present, Pax6 and Pax2 genes show structural differences
that reflect the changes that occurred in their coding sequences during
evolution. As well as the paired domain, Pax6 contains a homeodomain;
Pax2, however, has only part of the homeodomain and, in addition, an
octapeptide sequence accompanying the paired domain. Nevertheless, as
mentioned above, Drosophila Pax6 and Pax2 genes are
functionally exchangeable and can largely substitute for some of each other's
functions. The same seems to apply to the regulation of the DC5 enhancer in
Drosophila. Both Pax6 (ey and toy) and
D-Pax2 can activate this enhancer in vivo when they are co-expressed
with SoxN, but the main limitation to their real involvement in DC5
regulation is their expression pattern, meaning differences in their
regulatory elements.
In summary, we propose that after duplication of the PaxB-like ancestor, changes in the regulatory sequences determined which paralogous gene took over Crystallin regulation. Once the expression pattern of the duplicated genes diverged, changes in their coding sequences brought about the structural differences detected today, to better adjust each of the paralogous genes to its developmental role. According to our analysis, it seems that Crystallin regulation was taken over by Pax6 in vertebrates, whereas this function was retained by Pax2 in Drosophila.
Drosophila Crystallin genes under the control of the same regulatory circuit
The fact that lens-specificity of the DC5 fragment is retained in
Drosophila suggests that a similar mechanism could be responsible for
Crystallin regulation in the fruit fly. This is supported by the phenotype of
the Drosophila spapol mutant. In this mutant, as
previously mentioned, D-Pax2 expression is abolished in both the cone
cells and the primary pigment cells (Fu
and Noll, 1997). As a consequence, the hexagonal lattice of
ommatidia is severely disrupted, giving rise to a rough eye phenotype. In
spapol most of the cone cells and many primary pigment
cells are still present (Fu and Noll,
1997
) and retain the ability to secrete corneal lenses and
crystalline cones. However, these lens structures are frequently defective and
fused or display the blueberry-eye phenotype
(Fu and Noll, 1997
). We think
these lens defects are not a secondary effect of the disruption of the
ommatidium structure, but are probably due to the absence of D-Pax2
expression in the cone cells and primary pigment cells. Crystallin genes under
the putative control of D-Pax2 would fail to be expressed in this
situation, bringing about the lens defects observed in the Drosophila
spapol mutant.
As mentioned above, Drosophila Crystallin proteins are secreted by
cone cells and primary pigment cells, and accumulate on the top of the
ommatidium forming the corneal lens and the crystalline cone. Two-dimensional
gel electrophoresis has identified 14 different proteins in the crystalline
cone (Tomarev and Piatigorsky,
1996) and only three proteins in the corneal lens
(Komori et al., 1992
). The
most abundant protein in the corneal lens is Drosocrystallin, a 52 kDa protein
that contributes to lens development by providing the appropriate refractive
index to the corneal lens (Komori et al.,
1992
). Sequencing of the N-terminus end of Drosocrystallin allowed
the cloning of the drosocrystallin (dcy) gene and its
characterization as a member of the insect cuticular protein gene family
(Janssens and Gehring, 1999
).
Interestingly, dcy expression was detected in the primary pigment
cells, but not in the cone cells (Komori
et al., 1992
) (data not shown). As D-Pax2 is expressed in
both cell types, other regulatory differences between dcy and DC5
must exist to account for their distinct expression patterns. A 441 bp DNA
fragment, including the promoter region of dcy, was shown to be
sufficient to drive expression of a reporter gene into the primary pigment
cells (Janssens and Gehring,
1999
). We are currently dissecting this DNA fragment and
investigating its regulation to find similarities and differences with DC5
regulation.
The isolation and characterization of new Drosophila Crystallin
genes has been impaired by the heterogeneous nature of these genes. During
evolution, proteins with different enzymatic activities have been recruited
(or co-opted) to fulfil a Crystallin role, both in vertebrates and in
invertebrates. An event common to all these Crystallin co-options has been the
acquisition of highly lens-specific expression of the recruited proteins,
through changes in the regulatory regions of their genes
(Piatigorsky, 2003). The
results presented in this report suggest that the chicken DC5 enhancer might
be one of the genetic elements used throughout evolution to recruit new genes
into lens development. We are currently using this information to identify new
Drosophila Crystallin genes, and to find out whether their
lens-specific expression is achieved by regulatory elements similar to the
chicken DC5 enhancer.
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ACKNOWLEDGMENTS |
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