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First published online 12 April 2006
doi: 10.1242/dev.02328
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Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
* Author for correspondence (e-mail: duester{at}burnham.org)
Accepted 16 February 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Retinoic acid, Morphogenetic movements, Raldh1, Raldh2, Raldh3, Tbx5, Vax2, EphB2, ephrin B2, Eye, Optic cup, Retina, Perioptic mesenchyme, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Many ocular developmental defects are of unknown etiology and some may be
caused by environmental influences such as vitamin A deficiency in humans
(Hornby et al., 2002) or
animals (Wilson et al., 1953;
Marsh-Armstrong et al., 1994;
Dickman et al., 1997;
Reijntjes et al., 2005). One
important function of vitamin A (retinol) is to serve as the precursor of
retinoic acid (RA), an important developmental signaling molecule
(Duester, 2000). Some studies
suggest a connection between impaired retinol or RA function and developmental
eye defects in humans and mice. For instance, humans with missense mutations
in the gene encoding serum retinol binding protein have been shown to exhibit
retinal dystrophy (Seeliger et al.,
1999). Also, mice with reduced RA receptor signaling give rise to
offspring with ocular defects (Lohnes et
al., 1994).
RA serves as a ligand for three nuclear RA receptors (RAR) that bind DNA as
heterodimers with retinoid X receptors (RXR) and directly regulate gene
expression. Binding of all-trans-RA to the RAR component of RAR/RXR
heterodimers is necessary and sufficient to rescue signaling in RA-deficient
embryos, whereas the isomer 9-cis-RA (which can bind RXR) is unnecessary and
is undetectable under physiological conditions
(Mic et al., 2003). RARs are
expressed in overlapping patterns in the eye during development, and mice
carrying single null mutations of RARs have relatively normal eye development
except for RARß null mice, which exhibit a retrolenticular membrane in
the vitreous body (Ghyselinck et al.,
1997). Genetic elimination of two RARs results in microphthalmia,
ventral shortening of the retina, and abnormalities of the cornea, eyelids and
conjunctiva (Lohnes et al.,
1994). Thus, RARs mediate the ocular functions of vitamin A, as
the defects observed are essentially the same as those seen during gestational
vitamin A deficiency.
Based upon the expression patterns of RA-synthesizing enzymes, RA was
proposed to control dorsoventral patterning of the retina
(Wagner et al., 2000).
However, functional studies on these enzymes have not supported a role for RA
in the establishment of retinal dorsoventral patterning
(Fan et al., 2003;
Matt et al., 2005) or cell
fate determination (Mic et al.,
2004). A recent study in chick, using overexpression of
dominant-negative RA receptors has suggested that RA is needed to maintain
retinal dorsoventral patterning at later stages
(Sen et al., 2005), but this
has not been examined genetically. By contrast, RA has been clearly defined as
a signaling molecule needed for patterning of other regions of the central
nervous system, including the hindbrain and the spinal cord
(Maden, 2002). Although it is
clear that RA controls eye development, its mechanism may be different from
that of other eye signaling molecules that control patterning or cell fate
determination, such as fibroblast growth factor, which is released by the
surface ectoderm to stimulate neural retina differentiation
(Russell, 2003), or activin,
which is released by perioptic mesenchyme to stimulate retinal pigment
epithelium differentiation (Fuhrmann et
al., 2000). A recent genetic study in mice suggests that RA
controls eye development by acting within the neural crest-derived perioptic
mesenchyme (Matt et al.,
2005). The mechanism of RA action during eye development remains
unclear largely because of an incomplete understanding of the complex
spatiotemporal properties of RA synthesis in this organ and of the target
tissues upon which RA acts.
The first step of RA synthesis (the oxidation of retinol to retinaldehyde)
occurs ubiquitously through the action of several overlapping alcohol
dehydrogenases and short-chain dehydrogenase/reductases
(Molotkov et al., 2002). By
contrast, the second step of RA synthesis (oxidation of retinaldehyde to RA)
is limited to specific tissues by retinaldehyde dehydrogenases (i.e. RALDH1,
RALDH2, RALDH3) expressed in non-overlapping patterns in the developing mouse
eye (Mic et al., 2002;
Matt et al., 2005). Despite
their unique expression patterns, analyses of Raldh1, Raldh2 and
Raldh3 null mice have not resulted in clearly defined ocular defects,
probably because of the cell-nonautonomous action of RA generated by the
remaining enzymes (Niederreither et al.,
1999; Fan et al.,
2003; Dupé et al.,
2003; Mic et al.,
2004). Here, RALDH single and compound null mutant mice have been
analyzed to provide further insight into the role of RA in eye development,
and to sort out the individual contributions of each enzyme. We find that RA
signaling guides the morphogenetic movements of the retina and perioptic
mesenchyme, rather than playing a role in retinal dorsoventral patterning.
Furthermore, our studies reveal several paracrine mechanisms whereby RA is
released from RALDH-expressing cells and guides morphogenetic movements in
neighboring cells, thus demonstrating that RALDHs function
cell-nonautonomously in eye development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The Raldh3 gene targeting vector thus included a
loxP site upstream of exon 11, followed by exon 11, a
PGK-neo positive-selection gene downstream of exon 11 (transcribed in
same direction as Raldh3), and another loxP site just
downstream of PGK-neo. In order to generate a null allele, matings
were performed between the initial Raldh3-targeted chimeric males and
wild-type females expressing Cre recombinase in the germline in order to
remove exon 11 (i.e. about 600 bp of Raldh3) and PGK-neo in all
cells. PCR genotyping of tail or yolk sac DNA was performed using the primers
5'-GCCATAAAAGCTGGGGTGTCTG-3' (located in intron 10) and
5'-TGGATGGATGGATGGGTGATG-3' (located in intron 11) which produce a
wild-type product of 
950 bp and a mutant product of 350 bp (primer
annealing temperature was 63°C). Initial matings of
Raldh3+/- heterozygous null mice resulted in 70 adult
offspring with the following genotypes: 0 -/- (0%), 45 +/- (64%), 25 +/+
(36%). The absence of surviving homozygous mice indicated that the
Raldh3 knockout leads to a lethal phenotype. The description of
another Raldh3-/- mouse line indicated the existence of a
newborn lethal defect due to blockage of the nasal passages, leading to
respiratory stress at birth (Dupé et
al., 2003).
Generation of compound Raldh null embryos
Several Raldh mutant mice have been previously described,
including Raldh1-/- mice, which survive to adulthood
(Fan et al., 2003),
Raldh2-/- embryos, which exhibit midgestation lethality
(Mic et al., 2002), and
Raldh1-/-;Raldh2-/- double null
embryos, which also exhibit midgestation lethality
(Mic et al., 2004). Generation
of compound RALDH null mice was facilitated by independent assortment of the
three RALDH genes on separate chromosomes and by the ability to obtain adult
mice homozygous for the Raldh1 null allele. Matings were performed
between the above mice and Raldh3-/- mice to obtain
Raldh1-/-;Raldh3-/- and
Raldh2-/-;Raldh3-/- double null
embryos, as well as
Raldh1-/-;Raldh2-/-;Raldh3-/-
triple null embryos. Embryos derived from timed matings were genotyped by PCR
analysis of yolk sac DNA. Following mating, noon on the day of vaginal plug
detection was considered as embryonic day 0.5 (E0.5). Embryos were staged
according to somite number.
Rescue of eye defects with dietary RA supplementation
The rescue of Raldh2-/- embryos by maternal dietary RA
supplementation was performed similar to a previous description
(Mic et al., 2004), with an RA
dose demonstrated to be in the normal physiological range
(Mic et al., 2003). Briefly,
all-trans-RA (Sigma) was dissolved in corn oil and mixed with powdered mouse
chow to provide a final concentration of 0.1 mg/g for treatment from
E6.75-E8.5 (limited rescue), or 0.2 mg/g for treatment from E8.5-E14.5. In
some cases, embryos were analyzed when the mother was still on the
RA-supplemented diet, but in other cases the mother was returned to standard
mouse chow and embryos were analyzed at a later time point. Such food was
prepared fresh twice daily (morning and evening) and provided ad libitum.
Detection of retinoic acid
RA activity was detected as previously described in embryos carrying the
RARE-lacZ RA-reporter transgene, which places lacZ (encoding
ß-galactosidase) under the transcriptional control of a retinoic acid
response element (RARE) (Rossant et al.,
1991). Following genetic crosses of RARE-lacZ mice with
RALDH mutant mice, the RARE-lacZ transgene segregated independently
from Raldh1 and Raldh2, but not Raldh3, null
alleles, suggesting that RARE-lacZ is located close to
Raldh3 on mouse chromosome 7. In order to detect RARE-lacZ
expression in Raldh3-/- embryos, 20 male mice obtained
from RARE-lacZxRaldh3+/- crosses were
screened for a crossover event by mating to wild-type females, and one male
was identified that carried RARE-lacZ linked to the Raldh3
null allele. The RARElacZ-Raldh3 crossover male was also mated to
Raldh1 and Raldh2 mutant mice to enable the analysis of RA
activity in double and triple Raldh mutants. Stained embryos were
embedded in 3% agarose and sectioned at 50 µm with a vibratome.
In situ hybridization, proliferation and apoptosis assays
Whole-mount in situ hybridization was performed to detect expression of
Raldh1, Raldh2 and Raldh3, as described previously
(Mic et al., 2002). Probes for
Tbx5 (Chapman et al.,
1996) and Vax2 (Mui
et al., 2002) were also examined. Histological examination was
performed on paraffin-sectioned tissues stained with hematoxylin/eosin, as
previously described (Fan et al.,
2003). Cell proliferation assays were performed on paraffin tissue
sections by staining for histone-3 phosphorylation using antiphosphohistone-3
antibodies (Upstate Cell Signaling Solutions, Lake Placid, NY) as previously
reported (Molotkova et al.,
2005); cells counts were averages of six sections from comparable
ocular regions of two embryos (three sections per eye). Apoptosis was examined
in paraffin tissue sections using a TUNEL assay, as described
(Hyer et al., 2003).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Other
studies have recently shown that Raldh3-/- mice die at
birth as a result of nasal defects and exhibit a mild delay in ventral retina
growth (Dupé et al.,
2003). We generated an independent line of
Raldh3-/- mice, as well as
Raldh1-/-;Raldh3-/- double null mice,
and examined the effect on ocular development. Compared with wild-type and
Raldh1-/- embryos, which appeared identical at E14.5,
Raldh3-/- embryos initiated optic cup formation but
exhibited completely penetrant ocular defects consisting of excessive
thickening of the central neural retina, loss of the vitreous body, and a
prominent retrolenticular membrane between the retina and lens
(Fig. 1A-C; n=5 out of
5). Our Raldh3-/- null mutant exhibits more of a central
retina defect than that of another Raldh3-/- null mutant
that was reported to exhibit a shortening of the ventral retina
(Matt et al., 2005). This
could be due to strain differences, especially differences in how efficiently
Raldh2 acts in the two strains during early optic cup formation (see
below). Together, the findings from both strains suggest that RALDH3
synthesizes the RA required to complete optic cup and vitreous body
formation.
|
). We find
that loss of both Raldh1 and Raldh3 is required to observe
the defect because of functional redundancy. Another recent study also
observed excessive perioptic mesenchyme in
Raldh1-/-;Raldh3-/- double null
embryos (Matt et al., 2005).
As Raldh1 and Raldh3 are expressed in different regions of
the retina but not in the periocular mesenchyme
(Fig. 2G,I), this suggests that
RA synthesized in the retinal epithelium travels as a signal to the
surrounding mesenchyme to control morphogenetic movements of mesenchymal
ocular tissues.
The location of RA signaling activity in the developing eye at E10.5 was
examined in whole-mount wild-type and Raldh mutant mice carrying the
RARE-lacZ RA-reporter transgene
(Rossant et al., 1991).
Raldh3-/- embryos exhibited a large decrease in eye RA
activity compared with wild-type embryos, whereas
Raldh1-/- embryos exhibited only a modest decrease
(Fig. 1E-G).
Raldh3-/- embryos completely lost RA activity in the
ventral retina and olfactory pit, tissues that express Raldh3 but not
Raldh1 or Raldh2 at E10.5
(Fig. 2G-I). E10.5
Raldh1-/-;Raldh3-/- embryos carrying
RARE-lacZ exhibited almost a total loss of RA activity in the eye,
demonstrating that Raldh1 is primarily responsible for the RA
activity remaining in the eye of Raldh3-/- embryos
(Fig. 1H); residual RA activity
in Raldh1-/-;Raldh3-/- eyes is due to
RA synthesized by Raldh2 up to the point when its expression ends
near E9.75 (see below). These results confirm that RARE-lacZ is a
specific marker for eye RA signaling, as its expression is totally dependent
upon RA-synthesizing enzymes.
|
), we used
this method to rescue eye development in
Raldh1-/-;Raldh3-/- embryos. Following
RA treatment from E8.5-E14.5, we found that E14.5
Raldh1-/-;Raldh3-/- embryos exhibited
eye development that was very similar to normal with a vitreous body now
clearly observable, a neural retina of close to normal thickness, a diffuse
retrolenticular membrane, and normal perioptic mesenchyme growth
(Fig. 2A,B; n=3 out of
4). Examination of E11.5 rescued
Raldh1-/-;Raldh3-/- embryos carrying
RARE-lacZ revealed that maternally-derived RA had reached the neural
retina/vitreous body junction and dorsal perioptic mesenchyme
(Fig. 2D; n=4 out of
4), although such RA activity was significantly less than that observed in
Raldh1-/- littermates, where Raldh3 is generating
RA locally (Fig. 2C). As an
unrescued E11.5 Raldh1-/-;Raldh3-/-
eye completely lacked RA activity (Fig.
7E), maternal RA was responsible for the RA detected in rescued
mutants. Interestingly, the RA activity detected in the neural retina of
rescued Raldh1-/-;Raldh3-/- embryos
was not uniformly distributed, but was found along the periphery of the neural
retina closest to the vitreous body (Fig.
2D). It is also apparent that such RA activity is not detected in
the dorsal-most or ventral-most neural retina where Raldh1 and
Raldh3 are expressed (Fig.
2G,I) and where RA activity is normally found at high levels
(Fig. 2C), but instead is
observed in the central neural retina (Fig.
2D). Analysis of E12.5 rescued
Raldh1-/-;Raldh3-/- embryos
demonstrated high RA activity in the vitreous body and ventral perioptic
mesenchyme, and RA was more uniformly distributed throughout the central
neural retina but was still not detected in the dorsal-most or ventral-most
regions of the neural retina, which normally have highest levels of RA
activity (Fig. 2E,F;
n=2 out of 2). These results reveal that the targets of RA action
during eye development (inferred from the action of rescuing levels of
maternal RA) are distinct from the normal sites of RA synthesis.
Our observations with the RA-reporter transgene demonstrate that the low
dose of maternal dietary RA used to rescue
Raldh1-/-;Raldh3-/- eye development is
evidently not stimulating RA signaling in all portions of the eye. One
possible explanation for this observation is that RA may be preferentially
degraded in certain embryonic tissues, as has been observed for rhombomere 4
of the hindbrain, which induces a RA-degrading enzyme encoded by
Cyp26c1 to create an RA boundary
(Sirbu et al., 2005). Although
Cyp26a1 and Cyp26c1 function in RA degradation in the
central retina during late eye development (E15.5), no such role has been
found for earlier stages, and certainly no role has been found in the dorsal
or ventral retina at any stage (Sakai et
al., 2004). Another possibility for the selective appearance of RA
activity in rescued eyes is that incoming RA may be preferentially sequestered
by cellular RA-binding proteins (CRABP) that facilitate RA signaling
(Noy, 2000). In support of
this possibility, we found that Crabp1 is preferentially expressed in
the vitreous body (and weakly in perioptic mesenchyme), and that
Crabp2 is expressed in the dorsal and ventral perioptic mesenchyme
(Fig. 2J,K). These mesenchymal
sites of CRABP expression correspond to the regions in which the highest RA
activity is found during the rescue.
RA generated by Raldh1 controls perioptic mesenchyme invasion
Our experiments indicate that either Raldh1 or Raldh3
alone generates sufficient RA to control anterior invasion of perioptic
mesenchyme both dorsally and ventrally in Raldh3-/-
embryos. However, at E10.5, we detected RA activity in the dorsal but not
ventral perioptic mesenchyme of Raldh3-/- embryos
(Fig. 1G). This discrepancy was
resolved by examination at a later stage (E11.5), at which point
Raldh3-/- embryos did exhibit RA activity in both dorsal
and ventral mesenchyme (Fig.
3A,B). Thus, RALDH1 evidently generates increasing levels of RA in
the dorsal retina during development that are sufficient to reach the dorsal
and ventral perioptic mesenchyme by E11.5 to fulfill RA function in this
tissue.
RA is required for selective cell death in perioptic mesenchyme
In order to investigate RA function in the perioptic mesenchyme, we
examined the effect of a loss of RA on cell proliferation and apoptosis.
Ocular cell proliferation, examined in E11.5 embryos by the detection of
phosphohistone-3 (H3P), was not significantly different between
Raldh1-/- embryos that maintain relatively normal RA
activity and Raldh1-/-;Raldh3-/-
littermates that lose RA activity (Fig.
4A,B). The number of H3P-positive cells in
Raldh1-/- perioptic mesenchyme was 99±7.3, whereas
in Raldh1-/-;Raldh3-/- littermates the
number was 95±7.6 (n=6). For retina, the number of
H3P-positive cells in Raldh1-/- embryos was
26.3±2.8, whereas in
Raldh1-/-;Raldh3-/- littermates the
number was 27.5±1.1 (n=6). Thus, loss of RA does not effect
cell proliferation in either perioptic mesenchyme or retina. However, a loss
of RA does have an effect on apoptosis, as described in a recent study where
dorsal and ventral regions of apoptosis detected in the perioptic mesenchyme
of wild-type embryos were missing in
Raldh1-/-;Raldh3-/- embryos
(Matt et al., 2005). We found
that these dorsal and ventral regions of perioptic mesenchyme apoptosis were
still present in E11.5 Raldh1-/- embryos, but that almost
no apoptotic cells were detectable in
Raldh1-/-;Raldh3-/- littermates
(Fig. 4C,D; n=3 out of
3). The pattern of apoptosis in the retina did not appear to be significantly
different. These findings suggest that RA derived from either RALDH1 or RALDH3
functions in the perioptic mesenchyme to limit cell numbers by stimulating
apoptosis in selective regions.
RA is unnecessary to establish or maintain dorsoventral patterning of the retina
The unique expression patterns of Raldh1 in the dorsal retina and
Raldh3 in the ventral retina led to the hypothesis that RA generated
differentially in these two regions may somehow control dorsoventral
patterning of the retina (Wagner et al.,
2000). We examined this possibility by examining mutant embryos
for the expression of Tbx5 expressed dorsally
(Koshiba-Takeuchi et al.,
2000) and Vax2 expressed ventrally
(Mui et al., 2002;
Barbieri et al., 2002), both of
which play crucial roles in the establishment of retinal dorsoventral
patterning (McLaughlin et al.,
2003). We found that Tbx5 and Vax2 were
expressed in their correct retinal positions in E10.5
Raldh1-/-;Raldh3-/- embryos that lack
eye RA activity (Fig. 5A-C;
n=3). As it is possible that Raldh2 may have supplied RA for
dorsoventral patterning in
Raldh1-/-;Raldh3-/- embryos, we also
examined an
Raldh1-/-;Raldh2-/-;Raldh3-/-
triple mutant embryo at E10.5. In this embryo, Vax2 expression was
still detectable in the ventral optic vesicle, which had not invaginated to
form a complete optic cup (Fig.
5D). Previous studies had already shown that Tbx5 is
still expressed in Raldh2-/- embryos, and appears prior to
the ocular expression of Raldh1 or Raldh3
(Mic et al., 2002).
|
|
). However,
we found that ephrin B2 (Efnb2 - Mouse Genome Informatics) and
Ephb2 were still expressed in their correct locations in E14.5
Raldh1-/-;Raldh3-/- mouse embryos that
lack retinal RA activity (Fig.
5E-H; n=3). Thus, the previous studies that had relied
upon the introduction of a dominant-negative RA receptor may have been
artifactual (Sen et al.,
2005). Combined with our previous observations, these findings
suggest that RA generated by Raldh1 and Raldh3 in the neural
retina does not function cell-autonomously to establish or maintain
dorsoventral patterning of the neural retina, but instead functions
cell-nonautonomously to control survival of the adjacent perioptic
mesenchyme.
RA is necessary for ventral invagination of the optic cup
The observation that Raldh3-/- and
Raldh1-/-;Raldh3-/- embryos can
initiate optic cup formation indicates that RA is either unnecessary for
initial optic cup formation or that Raldh2 expressed during the optic
vesicle stage can provide RA for this function. We previously reported that
E10.5 Raldh2-/- embryos and
Raldh1-/-;Raldh2-/- embryos develop an
optic vesicle that fails to invaginate into an optic cup and fails to express
Raldh3, but that both of these defects could be rescued by maternal
dietary RA supplementation limited to E8.5 when Raldh2 expression is
first observed in the optic vesicle (Mic
et al., 2004). However, it is unclear whether the maternal RA
introduced during such a limited rescue had a direct effect on optic vesicle
invagination, which does not begin until E9.5, or was simply needed to remove
the impairment of trunk development caused by the loss of Raldh2,
which leads to a growth defect at E8.5 and lethality by E10.5
(Mic et al., 2002). In order
to resolve this issue, we examined limited-rescue
Raldh1-/-;Raldh2-/- embryos carrying
RARE-lacZ at E8.5 and found that RA activity was detected in the
trunk, but no activity was observed in the developing eye or anywhere in the
head (Fig. 7L). Thus, maternal
RA administered only to E8.5 does not stimulate RA signaling in the developing
eye, suggesting that maternal RA that enters the embryo during this
limited-rescue procedure acts posteriorly to rescue the survival of
Raldh1-/-;Raldh2-/- embryos, which
then allows optic vesicle development to continue to the stage when
Raldh3 expression begins.
In order to determine whether RA generated by Raldh3 in rescued Raldh2-/- embryos functions in initial optic cup formation, we generated Raldh2-/-;Raldh3-/- double null embryos. Raldh2-/-;Raldh3-/- embryos examined at E10.5-E11.5 following maternal dietary RA supplementation to E8.5 exhibited a failure in the ventral invagination of the optic vesicle that defines the junction between the ventral retina and optic stalk (Fig. 6A-D; n=3 out of 3). In these mutants, lens invagination had occurred, as well as dorsal invagination of the optic vesicle, which generates the junction between the retinal pigment epithelium (RPE) and the neural retina, thus resulting in an incomplete optic cup (Fig. 6A-D). As the ventral optic cup defect was still observed later at E11.5, this demonstrated a true blockage in ventral invagination rather than simply a delay (Fig. 6C,D). These results show that RA is required for ventral folding during initial optic cup formation in addition to the role described above for morphogenetic movements that limit neural retina thickness. Also, it is now clear that Raldh2 can provide this initial function in unrescued Raldh1-/-;Raldh3-/- embryos that develop an optic cup, and that Raldh3 can provide this function in limited-rescue Raldh1-/-;Raldh2-/- embryos.
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Contribution of each RALDH gene to eye development
The three RALDHs are expressed in unique tissues during mouse eye
development, and RA activity can be detected in those tissues in mice carrying
the RARE-lacZ RA-reporter transgene
(Wagner et al., 2000;
Mic et al., 2002). As RA
synthesized in one region of the developing eye can travel to other regions,
it has previously been impossible to determine the extent to which each enzyme
can supply RA to the eye and the importance of that RA. However, analysis of
RARE-lacZ expression in each of the RALDH double null mutants now
makes this possible, and comparison of the locations of such RA activity with
expression of the single remaining enzyme allows conclusions to be made
concerning the spatiotemporal relationship between sites of RA synthesis and
RA target tissues. The insights obtained from each RALDH double mutant are
described below.
Contribution of Raldh2
RA activity generated by Raldh2 in
Raldh1-/-;Raldh3-/- eyes was initially
detected throughout the optic vesicle epithelium (but not the surface
epithelium) prior to invagination, whereas when ventral optic vesicle
invagination was underway at E9.75 RA activity was much higher in the anterior
optic vesicle fated to become neural retina, and after optic cup formation at
E10.5 only a low level of RA activity remained in the most anterior and
central portion of the neural retina adjacent to the vitreous body
(Fig. 7A-D). At E11.5, no RA
was detectable in Raldh1-/-;Raldh3-/-
eyes (Fig. 7E). The location of
RA synthesis in Raldh1-/-;Raldh3-/-
eyes (determined by Raldh2 expression) was initially throughout the
optic vesicle epithelium prior to invagination
(Fig. 7F,G), matching well with
the RA activity at this stage (Fig.
7B). However, Raldh2 expression in the optic vesicle was
lost by E9.0 and replaced by expression in the mesenchyme adjacent to the
temporal portion of the optic vesicle from E9.0-E9.75
(Fig. 7H-K), and by E10.5 this
expression was also lost (Fig.
2H). Thus, during the stage when ventral optic vesicle
invagination occurs (E9.5-E9.75), RA is being synthesized by Raldh2
in the temporal mesenchyme and is traveling to the adjacent optic vesicle
where it acts. Our studies further suggest that early expression of
Raldh2 throughout the optic vesicle epithelium prior to invagination
is unnecessary for eye development. This is consistent with studies in chick
embryos demonstrating that Raldh2 is never expressed in the optic
vesicle epithelium but is expressed in the temporal mesenchyme
(Blentic et al., 2003). As
Raldh2 expression in mouse temporal mesenchyme ends before E10.5,
Raldh2 does not synthesize the RA needed for later morphogenetic
movements that limit neural retinal thickness and perioptic mesenchyme
growth.
|
). Studies in chick have demonstrated that optic cup
formation requires signaling from pre-lens ectoderm
(Hyer et al., 2003), but
evidently RA is not this signal as the optic cup develops in
Raldh3-/- embryos lacking RA synthesis in the pre-lens
ectoderm (Fig. 1C). Thus, the
early expression domain of Raldh3 in the surface ectoderm is an
insufficient source of RA for optic cup formation, making it clear that
Raldh3 expression in the RPE is the source of RA that acts on the
neural retina for ventral invagination. After RA from the RPE has stimulated
ventral invagination, Raldh3 expression initiates in the ventral
neural retina and RA travels throughout the optic cup and into the surrounding
mesenchyme, where it functions to limit perioptic mesenchyme growth
(Fig. 2C).
Contribution of Raldh1
RA activity was not detected in limited-rescue
Raldh2-/-;Raldh3-/- eyes at E9.5
(Fig. 7V,W), but low RA
activity generated by Raldh1 was observed at E10.0
(Fig. 7X) and high RA activity
was seen at E11.5 (Fig. 6D).
The location of RA synthesis in
Raldh2-/-;Raldh3-/- eyes inferred from
the Raldh1 expression pattern was limited to the dorsal neural retina
at both the optic vesicle stage, E9.5 (Fig.
7Y), and the optic cup stage, E10.5
(Fig. 2G). These findings
indicate that RA synthesized by Raldh1 accumulates too late to play a
role in ventral invagination of the optic vesicle or the control of neural
retina expansion. Our studies above have demonstrated that, later in
development, Raldh1 does not provide sufficient RA for vitreous body
morphogenesis, but does generate sufficient RA for the control of perioptic
mesenchyme growth both dorsally and ventrally
(Fig. 1C,D). As RALDH1 has a
10-fold lower enzyme activity for RA synthesis than RALDH2 or RALDH3
(Grün et al., 2000), the
relative inefficiency of RALDH1 as an RA source for eye development may be
understandable.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Tbx5 and Vax2 encode two transcription factors playing key
roles in establishing dorsoventral patterning of the retina
(Koshiba-Takeuchi et al.,
2000; Mui et al.,
2002; Barbieri et al.,
2002). However, we demonstrate here that E10.5
Raldh1-/-;Raldh3-/- embryos exhibit
correct dorsoventral expression of Tbx5 and Vax2 in the
retina. We further show that
Raldh1-/-;Raldh2-/-;Raldh3-/-
triple mutants that lack RA throughout the optic vesicle stage still express
Vax2, thus providing a completely conclusive genetic result. In
addition, our studies here indicate that RA function during eye development
does not commence until E9.5, after Tbx5 and Vax2 expression
has already been established (Chapman et
al., 1996; Mui et al.,
2002). Thus, genetic studies indicate that RA is not required for
the establishment of retinal dorsoventral patterning. A role for RA in
maintaining retinal dorsoventral patterning was suggested from studies in
chick embryos where dominant-negative RA receptors were found to interfere
with the expression of ephrin B2 and EPHB2. which function as dorsoventral
topographic guidance molecules (Sen et
al., 2005). However, our genetic studies demonstrate conclusively
that this is not the case, as
Raldh1-/-;Raldh3-/- embryos completely
lack RA activity in the retina, but maintain ephrin B2 and Ephb2
expression. We suggest that the introduction of dominant-negative RA receptors
may have wider effects on gene expression than was expected, possibly because
of the ability of RA receptors to heterodimerize with RXR receptors that
regulate not only RA signaling but also signaling by numerous other nuclear
receptors (Chawla et al.,
2001). RA signaling is also unnecessary for retinal determination,
as Raldh2-/- embryos lacking RA activity in the optic
vesicle still express early retinal determinants such as Pax6, Six3,
Rx and Mitf (Mic et al.,
2004). Instead, our findings here indicate that RA signaling
occurring within the retina itself is required to control the morphogenetic
movements that result in optic cup formation.
In addition to optic cup formation, neural tube closure represents another
major morphogenetic movement in the central nervous system. However, RA is
unnecessary for neural tube closure
(Wilson et al., 2003). Also,
RA has not previously been associated with neural morphogenetic movements,
although it has been reported to play a role in ventral retina formation
(Marsh-Armstrong et al., 1994;
Lohnes et al., 1994;
Dickman et al., 1997;
Reijntjes et al., 2005). Thus,
our studies define a novel role for RA in neural development, through its
ability to stimulate the morphogenetic movements needed for retinal
invagination. Our studies on
Raldh1-/-;Raldh2-/-;Raldh3-/-
embryos completely lacking eye RA activity demonstrate that RA is not required
for dorsal invagination of the optic vesicle that defines the junction between
the dorsal retina and pigment epithelium. However, our studies on these triple
mutants have revealed that RA is required for ventral invagination of the
optic vesicle that defines the junction between the ventral retina and optic
stalk. Also, our studies on Raldh3-/- single mutants
suggest that, as the neural retina expands during later stages of optic cup
formation, RA is required for the morphogenetic movements that restrict its
thickness and allow space for the vitreous body to form.
RA controls morphogenetic movements in retina and perioptic mesenchyme
This investigation has revealed the existence of two distinct phases of RA
signaling required for eye development, including an early phase for optic cup
formation and a late phase for anterior eye formation
(Fig. 8). In both cases, cells
expressing RALDH genes provide an RA signal that functions to control
morphogenetic movements in neighboring cells. Also, in both cases two RALDH
genes function as RA sources, providing functional redundancy.
During optic cup formation the sources of RA are Raldh3 expressed in the future RPE and Raldh2 expressed in mesenchyme on the temporal side of the optic vesicle; the RA target tissue is the neural retina. The outcome of this early phase of RA signaling is the stimulation of morphogenetic movements leading to ventral invagination of the optic vesicle to form a complete optic cup. Although Raldh2 provides sufficient RA to initiate ventral invagination of the neural retina, it alone is an incomplete source of RA, as its expression ends too early to provide the RA needed for proper distribution of neural retinal cells as the optic cup forms. However, Raldh3 expression continues and it alone can provide RA that is sufficient for both the initiation of ventral invagination and the proper distribution of neural retinal cells leading to formation of a normal vitreous body cavity. Our studies suggest that RA control of neural retina morphology does not involve the regulation of cell proliferation or apoptosis. Thus, RA may control cell movements within the retina.
|
), this
could be another source of RA that enters the perioptic mesenchyme. The
outcome of this late phase of RA signaling is the limitation of perioptic
mesenchyme morphogenetic movements that provide tissue contributing to the
corneal mesenchyme and eyelids (Cvekl and
Tamm, 2004). Raldh1 and Raldh3 can each alone
function as sufficient sources of RA for the control of perioptic mesenchyme
invasion. Our studies, and those of others
(Matt et al., 2005), suggest
that RA may perform this function by stimulating apoptosis in specific regions
of the perioptic mesenchyme, which would reduce cell numbers. However, a
restrictive effect of RA on mesenchymal cell migration cannot be ruled out.
The identification of perioptic mesenchyme as a target of RA synthesized in
ocular ectodermal tissues is consistent with other studies demonstrating the
need for perioptic mesenchyme to interact with retina and lens for proper eye
morphogenesis (Cvekl and Tamm,
2004).
RALDHs function cell-nonautonomously during eye development
Our studies with rescued RALDH mutant mice carrying an RA-reporter
transgene have revealed that the target of RA action changes during eye
morphogenesis. The initial target is the neural retina at the optic vesicle
stage, then the target switches to the perioptic mesenchyme after optic cup
formation. These targets are distinct from but adjacent to locations of RA
synthesis, thus demonstrating that RA does not need to function in the cells
that synthesize RA but instead functions in a paracrine fashion to guide the
morphogenetic movements of neighboring cells. Thus, all three RALDHs function
cell-nonautonomously. This has also been observed for posterior neural
development, where it has been determined that RA synthesized in the somitic
mesoderm by RALDH2 functions in the adjacent neuroectoderm but not in the
somites themselves (Molotkova et al.,
2005). Thus, in the neural tube, paracrine RA signaling stimulated
by RALDH2 occurs by mesenchymal to epithelial signaling. In the eye, we find
the existence of three paracrine RA signaling mechanisms: RALDH2 stimulates
mesenchymal to epithelial signaling for optic cup formation; RALDH3 stimulates
epithelial to epithelial signaling for optic cup formation; RALDH1 and RALDH3
both stimulate epithelial to mesenchymal signaling for anterior eye
formation.
Interestingly, for both neural tube and optic cup development, RALDH2
stimulates mesenchymal to epithelial signaling. This was not initially
suspected for optic cup development, as mouse Raldh2 is expressed
early in the optic vesicle epithelium just after its budding from the
forebrain, and only later exhibits expression in the mesenchyme adjacent to
the optic vesicle, just prior to optic cup formation. However, our studies
here on RA-rescued Raldh1-/-;Raldh2-/-
embryos carrying RARE-lacZ have demonstrated that a dose of RA
sufficient to rescue overall embryonic development and optic cup formation
does not stimulate RA signaling in the early optic vesicle epithelium. Thus,
Raldh2 expression in the early optic vesicle is unnecessary for optic
cup formation, a finding that is consistent with the observation that
Raldh2 is expressed in the temporal mesenchyme but not in the optic
vesicle epithelium of chick embryos
(Blentic et al., 2003). It is
unclear whether Raldh2 expression in the mouse optic vesicle serves
any function or whether it is an evolutionary relic.
RALDH3 provides sufficient RA to control morphogenetic movements
The investigations reported here illustrate the usefulness of compound
RALDH null mice in revealing the spatiotemporal role of RA signaling in
tissues where more than one RALDH contributes RA. Our findings make it clear
that each of the three RALDH genes contributes to eye morphogenesis.
Raldh1 can provide RA only for the control of perioptic mesenchyme
growth. Raldh2 is able to supply RA only for initial optic cup
formation, as its expression ends too early to provide RA for later
morphogenetic movements. Raldh3 alone can supply all of the RA needed
for eye morphogenetic movements in the mouse. Knowledge of the ocular RA
target tissues gained in these studies will facilitate the identification of
RA-regulated genes to further reveal the mechanisms by which RA controls
morphogenetic movements.
|
ACKNOWLEDGMENTS |
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