Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut Clinique de la Souris (ICS), CNRS/INSERM/ULP, Collège de France, BP10142, 67404 Illkirch Cedex, CU de Strasbourg, France
Author for correspondence (e-mail:
norbert{at}igbmc.u-strasbg.fr)
Accepted 8 August 2005
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SUMMARY |
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Key words: Retinoic acid receptor, Retinaldehyde dehydrogenase, Periocular mesenchyme, Somatic mutagenesis, Axenfeld-Rieger's syndrome
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Introduction |
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RA is not produced by all cells of the body at all stages of development,
but is generated in a unique spatio-temporal pattern
(Rossant et al., 1991). The
local conversion of vitamin A to RA involves two sequential oxidation steps:
(1) the generation of retinaldehyde from retinol, essentially carried out by
the ubiquitously expressed alcohol dehydrogenase type 3
(Molotkov et al., 2002
); and
(2) the synthesis of RA from retinaldehyde, catalyzed by retinaldehyde
dehydrogenases (RALDH) (Duester,
2000
). At least four isotypes (RALDH1 to RALDH4, encoded by mouse
genes Aldh1a1, Aldh1a2, Aldh1a3 and Aldh8a1, respectively)
are able to synthesize RA in vitro
(Duester, 2000
;
Lin et al., 2003
). RALDH1 is
less efficient than RALDH2 and RALDH3 at synthesizing all trans-RA
(Haselbeck et al., 1999
;
Grün et al., 2000
),
whereas RALDH4 is much more active for 9cis-RA synthesis
(Lin et al., 2003
). Only
RALDH1, RALDH2 and RALDH3 are expressed in the mouse developing eye, with
non-overlapping patterns. RALDH2 is transiently present at embryonic day (E)
8.5 in the optic vesicle (Wagner et al.,
2000
; Mic et al.,
2004
), while from E9.5 onwards RALDH1 and RALDH3 are mainly
detected in the dorsal and ventral fields of the retina, respectively
(McCaffery et al., 1999
;
Grün et al., 2000
;
Li et al., 2000
;
Mic et al., 2000
;
Suzuki et al., 2000
). Their
expression domains are separated by a stripe expressing the RA-degrading
enzymes CYP26A1 and CYP26C1 (Fujii et al.,
1997
; Sakai et al.,
2004
). This spatial arrangement of RALDH1, RALDH3, CYP26A1 and
CYP26C1 expression generates three areas along the dorsoventral (DV) axis of
the developing retina: a ventral one with high RA levels, a dorsal one
containing lower amounts of RA and an intermediate one free of RA; altogether,
these areas are thought to play a crucial role in DV patterning
(Wagner et al., 2000
;
Dräger et al., 2001
;
Peters and Cepko, 2002
;
Sakai et al., 2004
).
RALDH2, whose genetic ablation kills embryos at E9.5
(Niederreither et al., 1999;
Mic et al., 2002
), is required
between E8.5 and E9.5 for optic cup formation and for setting up the correct
expression of RALDH3 at later stages in the forming eye
(Mic et al., 2004
).
RALDH1-null mice are viable and do not exhibit ocular defects, even though
they lack RA-dependent activity in the dorsal retina and in the corresponding
axonal projections at E16.5 (Fan et al.,
2003
). RALDH3-null mice, which die at birth from respiratory
distress due to choanal atresia, display only discrete ocular malformations,
namely a retrolenticular membrane and a mild shortening of the ventral retina
(Dupé et al., 2003
).
Furthermore, neither RALDH1-null nor RALDH3-null mice exhibit an altered DV
patterning of the retina (Fan et al.,
2003
; Dupé et al.,
2003
). These observations raised the question as to whether the
loss of RALDH1 or RALDH3 could be functionally compensated by RALDH3 or
RALDH1, respectively, or by other RALDH proteins expressed in the eye region.
To gain further insight into the roles of RA signaling pathways in the
developing eye, we have analyzed the ocular phenotype of mutant mice (1)
lacking both RALDH1 and RALDH3, and (2) lacking RARß and RAR
selectively in the NCC-derived periocular mesenchyme (POM). We demonstrate
that RA produced in the neural retina, the retinal pigmented epithelium and
the corneal ectoderm by RALDH1 and RALDH3 acts in a paracrine manner to
selectively control the expression of several genes in POM cells, whereas it
is dispensable for the DV patterning of the retina.
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Materials and methods |
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Generation and genotyping of mice
To construct the Aldh1a1 targeting vector for homologous
recombination in embryonic stem (ES) cells (see
Fig. 1A), a 6 kb-long fragment
isolated from a 129/Sv mouse genomic DNA library and containing exons E7 and
E8, was inserted into pBluescript II SK+ (Stratagene). A loxP-flanked neomycin
resistance cassette (neo) was cloned into the BamHI site
located upstream of exon 8. Oligonucleotides
5'-ATAACTTCGTATAATGTATGCTATACGAAGTTATCCGCGG-3' and
5'-CCGCGGATAACTTCGTATAGCATACATTATACGAAGTTAT-3', containing loxP
and SacII sites, were then inserted 22 bp downstream of the
PpuMI site located on the 3' side of exon 8. This exon (amino
acid residues 249-283) encodes the aldehyde dehydrogenases glutamic acid
active site (E at position 268 in the LELGGKSP motif) in RALDH1. A
diphtheria toxin A (DT-A) expression cassette was inserted at the
3' side of the genomic DNA. This targeting vector was linearized with
KpnI and electroporated into ES cells. One clone, targeted as
expected (out of 146), was identified by Southern blot analysis (NO65, L3
allele, Aldh1a1+/L3). Transient transfection of NO65 ES
cells with a Cre-expressing plasmid allowed excision of the neo gene,
yielding cells bearing the conditional loxP-flanked L2 allele. One such clone
(NO65.23, L2 allele, Aldh1a1+/L2) was injected into
C57BL/6 blastocysts and the chimeras transmitted the conditional allele to
their germ line. Homozygous mice (Aldh1a1L2/L2) were
indistinguishable from their wild-type littermates. They were crossed with
CMV-Cre transgenic mice (Metzger
and Chambon, 2001), and the resulting mice bearing the excised
allele (Aldh1a1+/L) were identified by Southern
blot and PCR analysis. Tail DNA was genotyped by PCR using primers 1
(5'-GATTCCAGCAAACGGTAGGA-3') and 2
(5'-ACAGGATCAGGCATCAGGAG-3'; see
Fig. 1A) to amplify the
wild-type (871 bp long), the loxP-flanked L2 (1030 bp long), and the excised,
null, L (298 bp long) alleles. Conditions were 30 cycles,
with denaturation for 15 seconds at 92°C, annealing for 30 seconds at
61°C and elongation for 30 seconds at 72°C.
Western blot analysis
Cytosolic protein extracts (50 µg) were resolved on 12% SDS-PAGE gels
and blotted onto nitrocellulose membranes (Schleicher & Schuell). RALDH1
and RALDH3 were detected using purified IgG from rabbit polyclonal antisera
(gift from Dr J. L. Napoli) at a dilution of 1/500. Immunoreactions were
visualized using protein A coupled to horseradish peroxidase (dilution
1/10,000), followed by chemiluminescence according to the manufacturer's
protocol (Amersham).
Histology, staining and in situ RNA analysis
For histology, samples were fixed in Bouin's fluid for 5 days, embedded in
paraffin, serially sectioned, and stained with Groat's hematoxylin and
Mallory's trichrome. For ß-galactosidase activity detection, staining for
was performed as described (Rossant et
al., 1991). In situ RNA hybridization was carried out as described
(Dupé et al., 2003
).
The digoxigenin-labeled antisense riboprobes were synthesized using cDNA as
templates (references upon request). In situ hybridizations were performed on
serial histological sections along the entire anteroposterior axis of the
embryo head. All of the sections through the eye region (i.e. about 40) were
systematically analyzed. To ensure reproducibility, in situ hybridizations
were repeated on both eyes from at least two embryos of each genotype.
Terminal transferase-mediated dUTP-Nick-End-Labeling (TUNEL) was performed
using the Apoptag kit (Chemicon International) on 7 µm-thick serial frontal
sections from paraffin-embedded embryos. Note that the entire eye region,
which spans 40 sections, was analyzed and that TUNEL-positive cell clusters
(see Results) were detected on six to seven consecutive sections. Cell
proliferation was assessed on serial histological sections using the antibody
NCL-Ki-67p (Novocastra Laboratories) as described by the manufacturer. The
number of Ki-67-positive POM cell nuclei was determined by counting more than
2000 cells in at least four eyes of each genotype by the manufacturer.
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Results |
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RALDH1 and RALDH3 activities account for all RA synthesis in the developing eye region from E10.5 onwards
Vitamin A-deficient (VAD) fetuses, as well as fetuses carrying retinoid
receptor ablations, display severe ocular abnormalities that highlight the
crucial roles played by the RA-liganded RAR/RXR heterodimers in eye
development (see Introduction). By contrast, Aldh1a1-null mutants
have normal eyes, and fetuses lacking RALDH3 (hereafter designated
Aldh1a3-null fetuses) display mild ocular defects
(Dupé et al., 2003)
(see below). These observations raised the question as to whether the losses
of RALDH1 or RALDH3 could be functionally compensated by the other RALDH
proteins expressed in the eye region, even though such a functional
compensation appeared to be unlikely in the light of published data showing
the non-overlapping expression patterns of the genes encoding the three
RALDHs. Indeed, it has been reported that Aldh1a1 and
Aldh1a3 transcripts are confined to the dorsal and ventral retinal
fields, respectively (Li et al.,
2000
, Mic et al.,
2000
; Grün et al.,
2000
), whereas Aldh1a2 (encoding RALDH2) is transiently
expressed in the optic vesicle at E8.5 and thereafter in the periocular
mesenchyme (POM), but never in the neural retina
(Niederreither et al., 1997
;
Wagner et al., 2000
;
Mic et al., 2004
).
To determine whether a functional compensation between RALDHs may
nevertheless exist, we (1) re-investigated the expression profiles of
Aldh1a1, Aldh1a2 and Aldh1a3 during normal eye development,
and (2) analyzed the activity of the RARE-lacZ transgene in the
ocular region of Aldh1a1-null mutants, Aldh1a3-null mutants
and Aldh1a1/3-null mutants (lacking both RALDH1 and RALDH3;
see Materials and methods). At E10.5, E11.5 and E13.5, Aldh1a1 was
expressed in the dorsal retina and the lens as well as in the corneal ectoderm
(Fig. 2A,D,G), in accordance
with previous results (Li et al.,
2000; Suzuki et al.,
2000
; Mic et al.,
2000
; Grün et al.,
2000
). We additionally detected Aldh1a1 transcripts in
the temporal side of the ventral retina, not only at E13.5 as described
(Li et al., 2000
), but also at
E11.5 (Fig. 2D,G). This
discrepancy might be accounted for by a higher sensitivity of in situ
hybridizations on histological sections (present report) as compared with
whole mounts (Li et al.,
2000
). At E10.5, Aldh1a2 expression was absent in the eye
region (Fig. 2B), while, at
E11.5 and E13.5, it was localized in developing muscles present around the
eye, but was not detected in the POM per se
(Fig. 2E,H). At E10.5 and
E11.5, Aldh1a3 was expressed in the ventral retina
(Fig. 2C,F), as reported
(Grün et al., 2000
;
Mic et al., 2000
;
Li et al., 2000
;
Suzuki et al., 2000
). However,
we also detected Aldh1a3 expression in the peripheral portion of the
dorsal retina (arrowhead; Fig.
2C,F), throughout the retinal pigment epithelium, in the optic
nerve anlage and in the corneal ectoderm
(Fig. 2C,F). The expression of
Aldh1a3 was similar at E13.5, except in the retinal pigment
epithelium where the transcripts were no longer detected
(Fig. 2I).
Expression of the RARE-lacZ in the ocular region of E10.5 and
E11.5 wild-type fetuses was identical to that seen in Aldh1a1-null
mutants: a strong ß-galactosidase activity was detected at these
developmental stages in the retina, optic nerve, retinal pigment epithelium,
corneal ectoderm and POM, whereas a faint ß-galactosidase activity was
detected at the dorsal side of the lens (compare
Fig. 3A,E with 3B,F). At E13.5,
the transgene activity was undetectable in the dorsal retina of
Aldh1a1-null fetuses, except in its most peripheral portion (compare
Fig. 3I with 3J). These results
indicate that RALDH3 expressed at the peripheral portion of the dorsal retina,
the dorsal retinal pigment epithelium and/or the dorsal corneal ectoderm is
likely to compensate at E10.5 and E11.5 for the loss of RALDH1 by producing RA
that may diffuse to the dorsal retina. This compensation does not operate any
longer at E13.5. As for Aldh1a3-null mutants, the RA-dependent
ß-galactosidase activity was always maintained in the dorsal retina, the
dorsal corneal ectoderm and the dorsal POM
(Fig. 3C,G). These results
indicate that RALDH1 in the dorsal retina and corneal ectoderm probably
compensates for the loss of RALDH3, by producing RA that may diffuse to the
dorsal POM. By contrast, such a compensation does not occur in the retinal
pigment epithelium and in the ventral side of the eye, as ß-galactosidase
activity was abolished at E10.5 and E11.5 in the retinal pigment epithelium,
the ventral retina, the ventral corneal ectoderm and the ventral POM (compare
Fig. 3C,G with 3A,E). At E13.5,
a robust reporter activity appeared in the temporal side of the ventral retina
of Aldh1a3-null mutants (Fig.
3K), where Aldh1a1 transcripts were detected at this
stage (see Fig. 2G). Thus, at
E13.5, RALDH1 compensates for the loss of RALDH3 in the temporal side of the
ventral retina, but not in other ventral ocular tissues. Altogether, these
results indicate that the absence of eye malformations in
Aldh1a1-null mutants (Fan et al.,
2003) (and the present report), and the mild, ventral, ocular
defects of Aldh1a3-null fetuses
(Dupé et al., 2003
)
(and see below), may indeed be accounted for by a functional compensation
between RALDH1 and RALDH3.
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Absence of RALDH1 and RALDH3 yields severe eye abnormalities
The outcome of the lack of RA synthesis in the ventral region of the eye of
Aldh1a3-null mutants was investigated. At E14.5, these mutants
exhibited mild defects, including shortening of the ventral retina, ventral
rotation of the lens, persistence of the primary vitreous (retrolenticular
membrane) and thickening of the ventral periocular mesenchyme
(Dupé et al., 2003)
(see also below). Apart from the retrolenticular membrane, which is normally
present in E12.5 wild-type embryos
(Ghyselinck et al., 1997
),
these defects were much more apparent at E12.5 (compare
Fig. 4A with 4D). We have
previously suggested that similar ventral ocular defects in mutants lacking
either RXR
or RARß and RAR
could be related to the
thickened mesenchyme interposed between the retina and the ectoderm
(Mark et al., 1998
). To
investigate the cause of the POM thickening in Aldh1a3-null mutants,
apoptosis was analyzed at E10.5 (Fig.
4B) and E11.5 (not shown) by performing TUNEL assays on serial
histological sections along the anteroposterior axis of the eye region. In the
POM of wild-type embryos, we identified two clusters of TUNEL-positive cells,
a dorsal one located in the posterior region and a ventral one located in the
anterior region, both at E10.5 (n=3)
(Fig. 4B) and at E11.5 (see
below, Fig. 7A,D). These two
clusters may be instrumental to the physiological remodelling of the POM,
especially because TUNEL-positive cells represent only a small fraction of the
cells committed toward apoptosis (Gavrieli
et al., 1992
). In Aldh1a3-null eyes, the ventral cluster
was not detected at E10.5 (n=2) (brackets,
Fig. 4E), and was of a reduced
size at E11.5 (n=2) (not shown). It has been proposed that the
expression level of Eya2 correlates with programmed cell-death during
eye development (Xu et al.,
1997
; Clark et al.,
2002
). In E11.5 wild-type embryos, Eya2 was strongly
expressed in the POM, both in the ventral and the dorsal sides
(Fig. 4C; and data not shown,
see below). In E11.5 Aldh1a3-null mutants (n=1),
Eya2 expression was specifically reduced in the ventral POM
(Fig. 4F). This observation
indicates that the decreased programmed cell-death in the ventral POM of
Aldh1a3-null mutants, caused by impairment of RA synthesis in the
ventral eye region, correlates with a decreased Eya2 expression in
ventral POM cells.
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The periocular mesenchyme is the primary target of RA action
The eye defects of Aldh1a1/3-null fetuses are similar to
those observed in mutant fetuses lacking both RARß and RAR
(Ghyselinck et al., 1997
).
RARß and RAR
, which both act in a heterodimer with RXR
(Kastner et al., 1994
;
Kastner et al., 1997
), are
normally expressed in the cells surrounding the developing optic cup (i.e. in
the POM), but not in the retina
(Ghyselinck et al., 1997
;
Mori et al., 2001
). Therefore
RA synthesized by RALDH1 and RALDH3 in the neural retina may control eye
development through the activation of RXR
/RARß and
RXR
/RAR
heterodimers in POM cells. To test this possibility, we
performed Cre-mediated somatic ablation of the Rarb and Rarg
genes (Metzger and Chambon,
2001
) in POM cells, which largely originate from the neural crest
(Johnston et al., 1979
;
Trainor and Tam, 1995
). To
this end, we used a transgenic line (Wnt1-Cretg/0),
expressing Cre under the control of the Wnt1 promoter
(Danielian et al., 1998
),
which is active in neural crest cells (NCCs). We also used a mouse line
bearing a conditional reporter transgene (R26Rtg/0), which
expresses ß-galactosidase only upon Cre-mediated recombination of a
loxP-flanked (floxed) intervening DNA sequence
(Soriano, 1999
). This reporter
continues to be expressed in all progeny of the NCCs that have undergone
recombination, even though they are no longer expressing Cre
(Jiang et al., 2000
). In E10.5
Wnt1-Cretg/0/R26Rtg/0 fetuses
(hereafter designated WTNCC+/+ lacZ), reporter activity was
detected in POM cells (Fig.
5M), including those forming the stroma of the cornea (data not
shown), but not in other ocular structures. Therefore, Wnt1-driven
expression of Cre allows efficient recombination in POM cells.
Mice in which both alleles of the genes encoding RARß and RAR
are floxed (Rarb/gL2/L2 mice) were then crossed with
Wnt1-Cretg/0R26Rtg/0 transgenic mice,
to generate fetuses in which RARß and RAR
ablation was restricted
to NCCs and to their derivatives, including the POM (hereafter designated
Rarb/gNCC/ lacZ mutants). Importantly, the
pattern of ß-galactosidase activity identifying NCC derivatives was
identical in E10.5 Rarb/gNCC/ lacZ and
WTNCC+/+ lacZ fetuses, demonstrating that ablation of RARß and
RAR
in NCCs did not alter their migration from the neurectoderm into
the periocular region (compare Fig. 5N with
5M). Similarly to Aldh1a1/3-null
(Fig. 5I) and
Rarb/g-null mutants (Ghyselinck
et al., 1997
), E14.5 Rarb/gNCC/
lacZ fetuses (n=2) exhibited an abnormal, thick layer of
mesenchyme replacing the eyelids and the cornea
(Fig. 5J), and a small and
abnormal conjunctival sac (Fig.
5J), as well as a severe shortening of the ventral retina, ventral
rotation of the lens, and persistence and hyperplasia of the primary vitreous
body (Fig. 5J). These results
demonstrate that selective ablation of RARß and RAR
in the POM
yields eye defects identical to those displayed by mutants lacking RALDH1 and
RALDH3.
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|
Ablation of Rarb and Rarg in neural crest cells impairs apoptosis and alters gene expression in periocular mesenchymal cells
In the POM of Rarb/gNCC/ lacZ eyes
(n=2), the number of Ki-67-positive cells (406±34) was similar
to that in wild-type eyes (see above) (compare
Fig. 6A with 6C). Therefore, as
in Aldh1a1/3-null mutants, cell proliferation in the POM appeared to
be unaffected by the ablation of Rarb and Rarg in NCCs.
Moreover, and similarly to Aldh1a1/3-null mutants, the normal ventral
and dorsal clusters of apoptotic POM cells
(Fig. 8A,B; and see above) were
not detected in Rarb/gNCC/ lacZ mutant eyes
(n=4; Fig. 8C,D).
Along these lines, expression of Eya2 appeared to be reduced in the
POM in Rarb/gNCC/ lacZ mutant eyes
(n=2; compare Fig. 8E with
8G). Finally, it is noteworthy that expression of both
Pitx2 (data not shown) and Foxc1 appeared to be also
decreased in the POM in Rarb/gNCC/ lacZ
mutant eyes (n=4; compare Fig. 8F
with 8H). Therefore, similar cellular and molecular defects are
generated in the POM upon the ablation of RALDH1 and RALDH3, or of
Rarb and Rarg.
Dorsoventral patterning of the retina does not depend upon RA
It has been proposed that RA is important for the dorsoventral (DV)
patterning of the retina (McCaffery et
al., 1999; Peters and Cepko,
2002
). Thus, we analyzed whether impairing RA synthesis, through
the combined ablation of RALDH1 and RALDH3, alters the DV patterning of the
retina. The identity of progenitor cells along the retina DV axis is specified
by the expression of Tbx5, Vax2 and Pax2 genes (reviewed by
McLaughlin et al., 2003
),
while the stereotyped distribution of apoptotic cells during retina
development provides a reliable landmark of DV axis patterning
(Laemle et al., 1999
). The
distribution patterns of Tbx5 and Vax2 transcripts at E10.5
(Fig. 9A-D), and of
Pax2 transcripts at E11.5 (compare
Fig. 9E with 9F), as well as
the distribution of TUNEL-positive cells at E10.5 (compare
Fig. 9G with 9H), were
undistinguishable in Aldh1a1/3-null and wild-type fetuses.
Altogether, our results show that impairing RA synthesis in the retina from
E10.5 onwards does not alter the formation of its DV axis.
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Discussion |
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|
|
Different mechanisms, including cell proliferation, apoptosis and the
extent of extracellular matrix deposition, may control the thickness of the
POM. On the one hand, we show that cell proliferation is not affected upon
ablation of either RALDH1 and RALDH3, or RARß and RAR. On the
other hand, we show, in POM cells of both Aldh1a1/3-null and
Rarb/gNCC/ lacZ mutants, a decreased
expression of Eya2 that may account for the impaired apoptosis
(Xu et al., 1997
;
Clark et al., 2002
), and thus
for the thickening of the POM, especially as our result also indicates that
eye morphogenesis in the wild-type situation is accompanied by cell-death in
the POM. However, we cannot exclude the possibility that other mechanisms,
such as changes in the composition of the extracellular matrix, may also
participate in the thickening of the POM. In any event, we propose that RA
synthesized by RALDH1 and RALDH3 in the retina, the retinal pigment epithelium
and/or the corneal ectoderm activates RXR
/RARß and
RXR
/RAR
heterodimers in POM cells, which in turn control the
Eya2-related programmed cell death
(Fig. 10).
We also found that losses of RALDH1 and RALDH3, and the selective ablation
of Rarb and Rarg in NCCs, apparently decrease, in POM cells,
the expression of Pitx2 and Foxc1, which code for
transcription factors involved in eye morphogenesis (Cvelk and Tamm, 2004). RA
treatment has been shown to induce Pixt2 expression in embryonic stem
cells (Lindberg et al., 1998).
Furthermore, mutations in PITX2 and FOXC1 generate the human
Axenfeld-Rieger's syndrome, which includes ocular anterior segment defects
(reviewed by Lines et al.,
2002
). Haploinsufficiency or deletion of either Pitx2 or
Foxc1 in the mouse (Kume et al.,
1998
; Kitamura et al.,
1999
) induces an anterior segment dysgenesis, characterized by an
absence of the anterior chamber, a thickening of the mesenchymal component of
the cornea, an agenesis of the iris, the persistence of the primary vitreous
body and a ventral rotation of the eye, all of which closely resemble the
ocular defects displayed by Aldh1a1/3-null and
Rarb/gNCC/ lacZ mutant mice. Thus, the
malformations induced by the loss of RALDH1 and RALDH3, and the ablation of
Rarb and Rarg, most probably result from a reduced
Pitx2 and Foxc1 expression. We propose therefore that RA
synthesized by RALDH1 and RALDH3 in the retina, the retinal pigment epithelium
and/or the corneal ectoderm activates RXR
/RARß and
RXR
/RAR
heterodimers in POM cells, which in turn control the
expression of Pitx2 and Foxc1 required for normal
morphogenesis of the eye anterior segment
(Fig. 10).
We found that fetuses lacking RALDH1 and RALDH3 in the retina, the retinal
pigment epithelium and the corneal ectoderm (Aldh1a1/3-null mutants),
or selectively missing RARß and RAR in the POM
(Rarb/gNCC/ lacZ mutants), displayed an
identical shortening of the ventral retina. Thus, it appears that RA
synthesized by RALDH1 and RALDH3 activates RXR
/RARß and
RXR
/RAR
heterodimers in POM cells, which in turn may produce a
`paracrine' signal necessary for the proper growth of the ventral retina
(Fig. 10).
|
A sequential role for RALDH1, RALDH2 and RALDH3 during eye morphogenesis
From E9.0 to E16.5, eye morphogenesis involves the coordinated development
of forebrain neuroectoderm, surface ectoderm and NCC-derived mesenchyme
(Johnston et al., 1979). The
panel of ocular malformations induced by vitamin A-deficiency
(Warkany and Schraffenberger,
1946
; Wilson et al.,
1953
; Dickman et al.,
1997
), or upon impairment of RA signaling
(Kastner et al., 1994
;
Kastner et al., 1997
;
Lohnes et al., 1994
;
Ghyselinck et al., 1997
;
Mascrez et al., 1998
),
indicates that RA-liganded RAR/RXR heterodimers are required at all stages of
eye development. Mutant fetuses lacking RALDH2 fail to form an optic cup
(Mic et al., 2004
), but their
rescue through maternal administration of RA before E9.5 prevents the
occurrence of ocular malformations and restores a normal expression pattern of
RARE-lacZ in the eye (Mic et al.,
2002
; Niederreither et al.,
2002
), suggesting an involvement of RALDH2 in eye morphogenesis
until E9.5, but not at later stages. In the present study, we show that (1)
the activity of RARE-lacZ is abolished in the eye region of
Aldh1a1/3-null fetuses from E10.5 to E13.5, and (2) the ocular
defects reported for Aldh1a1/3-null mutants all reflect a loss of RA
signaling occurring from E10.5 onwards. Altogether, these data indicate that
RALDH1 and RALDH3, expressed in the retina, retinal pigment epithelium and
corneal ectoderm, are actually the only RA-synthesizing enzymes required in
the eye region from E10.5 onwards.
The spectrum of ocular defects in Aldh1a1/3-null and
Rarb/gNCC/ lacZ mutants recapitulates that
induced by vitamin A-deficiency and/or loss-of-function mutations of RAR and
RXR genes, with the exceptions of open eyelids and agenesis of the lens
(Lohnes et al., 1994;
Ghyselinck et al., 1997
;
Kastner et al., 1997
;
Mascrez et al., 1998
). The
RA-degrading enzyme CYP26B1 is expressed in the mesenchyme underlying the
palpebral fissure just prior to eyelid fusion
(Abu-Abed et al., 2002
), and
Cyp26b1-null mutants display open eyes at birth
(Yashiro et al., 2004
).
Therefore, eyelid fusion is likely to require unliganded RA-receptors, but not
RALDH1 and RALDH3. Agenesis of the lens in Rara/Rarg-null mutants has
been ascribed to a default in lens induction, a process that depends upon
interactions between the optic vesicle and the surface ectoderm, and which
occurs before E10.5 (Lohnes et al.,
1994
). A recent study shows that RA synthesized by RALDH2 in the
optic vesicle between E8.5 and E9.5 is actually required for optic cup and
lens formation (Mic et al.,
2004
). Altogether, these data add to the notion that RALDH2 is the
RA-synthesizing enzyme needed during the early steps of ocular morphogenesis
(before E9.5), while RALDH3 and RALDH1 are required for the subsequent steps
of eye development (from E10.5 onward).
RA is not required for the dorsoventral patterning of the retina
During early eye morphogenesis, RALDH1 and RALDH3 generate RA-rich regions
in the dorsal and ventral fields of the retina, separated by a RA-depleted
zone expressing the catabolizing enzymes CYP26A1 and CYP26C1
(Fujii et al., 1997;
McCaffery et al., 1999
;
Grün et al., 2000
;
Li et al., 2000
;
Mic et al., 2000
;
Suzuki et al., 2000
;
Sakai et al., 2004
). Several
authors have proposed that the combined activities of these enzymes set up a
RA gradient in the retina that is important for its dorsoventral (DV)
patterning (McCaffery et al.,
1999
; Peters and Cepko,
2002
). Neither the ablation of RALDH1
(Fan et al., 2003
) nor that of
RALDH3 (Dupé et al.,
2003
) alters the retina DV axis. However, the actual role of these
two enzymes in setting this DV axis could have been masked by a functional,
reciprocal compensation of RALDH1 by RALDH3. In the present study, we show
that impairing RA synthesis in the retina from E10.5 onwards, through RALDH1
and RALDH3 ablation, does not alter the formation of its DV axis. Therefore,
contrary to previous expectations
(McCaffery et al., 1999
;
Peters and Cepko, 2002
), RA is
actually not required to set up the DV axis of the retina. However, our
results do not exclude a role for RA in setting up the other retinal axes, or
in retinal histogenesis (Grondona et al.,
1996
).
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ACKNOWLEDGMENTS |
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Footnotes |
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