1 Neurobiology and Animal Physiology, Institute of Biology I, University of
Freiburg, D-79104 Freiburg, Germany
2 Developmental Biology, Institute of Biology I, University of Freiburg, D-79104
Freiburg, Germany
* Author for correspondence (e-mail: johannes.von.lintig{at}biologie.uni-freiburg.de)
Accepted 13 February 2003
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SUMMARY |
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Key words: Zebrafish, Carotenoid conversion, Vitamin A, ß, ß-carotene-15, 15'-oxygenase, Retinoic acid, Neural crest, Craniofacial skeleton, Eyes, Pectoral fins
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INTRODUCTION |
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Retinoid signaling depends on the availability of maternal sources for
vitamin A and is controlled through the spatially and temporally regulated
expression of enzymes involved in RA synthesis and catabolism (reviewed by
Ross et al., 2000). Retinal
dehydrogenases (Raldhs) have been clearly identified as the enzymatic
activities catalyzing the final step in RA synthesis. Targeted inactivation of
Raldh2 in mouse, as well as mutations in raldh2 in
zebrafish, both generate phenotypes mimicking most features of an early onset
embryonic VAD (Niederreither et al.,
1999
; Niederreither et al.,
2000
; Begemann et al.,
2001
; Grandel et al.,
2002
; Mic et al.,
2002
). During organogenesis, besides raldh2, two
additional retinal dehydrogenases, raldh1 and raldh3, are
expressed at distinct sites where they may contribute to the generation of
local RA signals (Li et al.,
2000
; Mic et al.,
2002
; Niederreither et al.,
2002
). It is assumed that retinal synthesis from retinol
constitutes the upstream step of the RA-generating pathway. In the mammalian
embryo, retinol is provided from the maternal circulation via the placenta,
whereas avian, reptilian, amphibian and fish embryos use retinoid stores in
the egg yolk. A multitude of retinoid metabolizing enzymes were characterized
for retinol to retinal conversion in vitro and can be assigned to two enzyme
families: alcohol dehydrogenases (ADH) and short chain
dehydrogenases/reductases (SDR) (for a review, see
Duester, 2000
). Although, so
far, no genetic evidence exists that any of these enzymes may act in vivo
upstream to the different types of Raldh, recent results suggest that Adh3, an
ubiquitously expressed retinol-oxidizing enzyme, is needed for mouse
development, as loss of its function leads to postnatal lethality
(Molotkov et al., 2002
).
Besides retinoids, provitamin A-carotenoids are present in the mammalian
circulation and in the vertebrate egg yolk. The enzymatic conversion of these
compounds results in the generation of retinal, the direct precursor of RA
(Olson and Hayaishi, 1965;
Goodman and Huang, 1965
). A
possible role of this alternative source for retinoids has not yet been
addressed, as the molecular nature of this enzyme has remained elusive for a
long time. Our recent research led to the molecular identification and
functional characterization of the gene encoding the key enzyme in provitamin
A conversion, a ß,ß-carotene-15,15'-oxygenase (bcox)
in Drosophila (von Lintig and
Vogt, 2000
; von Lintig et al.,
2001
). Vertebrate orthologs were then identified based on sequence
identity from various species, including human
(Wyss et al., 2000
;
Redmond et al., 2001
;
Kiefer et al., 2001
;
Paik et al., 2001
;
Yan et al., 2001
).
Building on this work, the present study aimed to elucidate the impact of provitamin A conversion on embryonic development in zebrafish (Danio rerio). We cloned the cDNA of the vitamin A-generating enzyme (bcox) and demonstrated the presence of its substrate ß-carotene in zebrafish egg yolk by HPLC analyses. We then showed that bcox is expressed in clearly defined spatial compartments and translated into protein in the zebrafish embryo. To test whether there is an actual requirement for provitamin A conversion during embryonic development, we performed targeted gene knockdown experiments using morpholino antisense oligonucleotides (MO). The bcox morphants developed abnormalities of the craniofacial skeleton, pectoral fins and eyes, which are impairments well known from VAD embryos. Analyses of changes in gene expression in bcox morphants revealed specific defects in patterning and differentiation during several distinct RA-dependent developmental processes, thus providing the first evidence that carotenoids are indispensable as precursors for RA signaling in a vertebrate.
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MATERIALS AND METHODS |
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The zebrafish mobm819 mutant is a null allele of
ap2 and details of the phenotype are available (J.H., A.
Barrallo, A. K. Ettl, E. Knapik and W.D., unpublished).
Cartilage of zebrafish larvae was stained with Alcian Blue as described by
Neuhauss et al. (Neuhauss et al.,
1996). Microtome sections (3 µm) of plastic-embedded 5-day-old
larvae were mounted on poly-L-lysine coated slides, air dried at 60°C,
stained with Richardson's solution (1% azure, 1% Methylene Blue, 1% borax in
deionized water), overlaid with DPX (Merck, Germany) and coverslipped.
Cloning and functional characterization of bcox
Total RNA from liver of adult zebrafish was isolated with the high pure RNA
isolation kit (Roche Molecular Biochemicals) according to the manufacturer's
protocol. For cloning a full-length cDNA encoding the putative zebrafish
ß-carotene-15,15'-oxgenase, RACE-PCRs were performed using a
5'/3' RACE kit (Roche Molecular Biochemicals). For 3'-RACE,
reverse transcription was performed with 500 ng of total RNA isolated from
liver, an oligo-d(T)-anchor primer
(5'-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV-3') and Superscript
reverse transcriptase II (Life Technologies). For PCR, the Expand PCR system
(Roche Molecular Biochemicals), an anchor primer and a specific primer,
5'-GGTGCAGGGAACACTAATACG-3'derived from a published EST-fragment
(GenBank Accession Number AW128477) were used. For 5'-RACE, reverse
transcription was performed with 500 ng of total RNA from liver, a specific
primer 5'-CTCGAATGGGTGCAGGGAACA-3' derived from the EST fragment,
and Superscript reverse transcriptase (Life Technologies, Inc.). For PCR, the
Expand PCR system (Roche Molecular Biochemicals), the specific primer
5'-CTCGAATGGGTGCAGGGAACA-3' and an anchor primer were used. The
PCR fragments were cloned in the vector pCR2.1 TOPO (Invitrogen) and subjected
to sequence analyses. The full-length cDNA sequence of bcox was
deposited with GenBank (Accession Number AJ290390).
The entire coding region of the bcox cDNA was amplified by RT-PCR.
For reverse transcription 500 ng total RNA was incubated in the presence of a
polydT17 primer and Superscript II (Life Technologies). For PCR,
the Expand PCR system (Roche Molecular Biochemicals), and the primers
5'-ATGCAGTACGACTATGGCAAA-3' and
5'-GCTGCTGCCTGGTATGAAGTA-3' were used. The PCR product was ligated
into the vector pCR-BluntII-TOPO (Invitrogen) and transformed in a
ß-carotene synthesizing and accumulating E. coli strain. Tests
for enzymatic activity of the Bcox protein were performed as previously
described (von Lintig and Vogt,
2000).
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed as described by Hauptmann
and Gerster (Hauptmann and Gerster,
1994). bcox was cloned into the vector pBluescript SK
(Stratagene) and antisense and sense RNA probes were synthesized with the T7
RNA polymerase and T3 RNA polymerase, respectively. Additional RNA probes used
for in situ hybridization experiments were shh
(Krauss et al., 1993
),
efna4 (Xu et al.,
1996
), hoxb4a (Prince
et al., 1998
), fgf8
(Reifers et al., 1998
),
myod (Weinberg et al.,
1996
), krx20 (egr2 Zebrafish Information
Network) (Oxtoby and Jowett,
1993
), dlx2 (Akimenko
et al., 1994
), pax6.1 (pax6a Zebrafish
Information Network) (Püschel et al.,
1992
), pax2.1 (pax2a Zebrafish
Information Network) (Krauss et al.,
1991
), ap2
(tfap2a Zebrafish
Information Network) (J.H., A. Barrallo, A. K. Ettl, E. Knapik and W.D.,
unpublished) and val (Moens et
al., 1998
). The RNA probes were generated with the Dig RNA
labeling kit (Roche Molecular Biochemicals) according to the manufacturer's
protocol.
Injections of morpholino oligonucleotides and synthetic bcox
mRNAs
For targeted knockdown of the bcox protein, two antisense
morpholino oligonucleotides (GeneTools, LLC) were used that covered either
nucleotides +1 to +25 (bcox-MO;
5'-TGTTTTTGCCATAGTCGTACTGCAT-3') or 24 to 1
(bcox-MOb; 5'-CCTTTCAGATGCTTTCTTCAAGTTC-3') of the
bcox mRNA. For control injections, a standard control morpholino
oligonucleotide [co-MO; 5'-CCTCTTACCTCAGTTACAATTTATA-3' (Gene
Tools, LLC)] was used. The MOs were dissolved in 0.3x Danieau's solution
[1x Danieau: 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM
Ca(NO3)2, 5 mM HEPES, pH 7.6] to obtain a stock
concentration of 1 mM (8.43 mg/ml). The stock solutions were diluted to 6, 3
and 0.5 mg/ml. The injected volume was 3 nl, corresponding to 18, 9 and
1.5 ng morpholino oligonucleotide per egg, respectively. The bcox
cDNA was cloned into the vector pCS2+ and RNA was synthesized in vitro using
the mMESSAGEmMACHINE kit according to the manufacturer's protocol (Ambion
Europe, UK). Co-injection of various amounts of the bcox mRNA was
used to control the specificity of bcox-MO. For rescue experiments,
various amounts of this modified bcox mRNA were co-injected with 9 ng
of bcox-MO. In addition, the targeted knockdown of the bcox
protein was monitored using western blot analyses.
Retinoic acid and citral treatments
bcox morphants and wild-type embryos were exposed to
109 M RA in egg water diluted from a stock of
3x101 M all-trans retinoic acid in dimethyl sulphoxide
(DMSO) (Sigma, Germany). The start and duration of incubation are indicated
for the respective experiments. Incubations were carried out in the dark and
embryos were fixed at desired stages. At the three-somite stage, wild-type
embryos were incubated in egg water containing 250 µM citral (Roth,
Germany) for 1 hour. After treatment, embryos were washed three times for 10
minutes in egg water to remove citral and fixed at desired stages.
Western blot analysis
For western blot analyses we used a polyclonal serum raised against the
mouse Bcox (Kiefer et al.,
2001). We first verified the crossreaction of the serum with
zebrafish Bcox expressed in E. coli. For western blot analyses with
zebrafish embryos, four dechorionized embryos were directly homogenized in SDS
lysis buffer (63 mM Tris HCl, pH 6.8, 10% glycerol, 5% 2-mercaptoethanol, 3.5%
sodium dodecyl sulfate) and subjected to SDS-PAGE according to Westerfield
(Westerfield, 1994
).
Alternatively, 100 dechorionized embryos were homogenized in 200 µl Tricine
buffer (50 mM Tricine, 100 mM NaCl, pH 7.5) using a loose-fitting glass
potter. Protein was harvested (50 µg) using StrataClean resin (Stratagene)
and subjected to western blot analysis. Immunostaining was carried with the
serum diluted 1:500 and the ECL system according to the manufacturer's
protocol (Amersham Pharmacia Biotech).
HPLC analysis of carotenoids and retinoids of zebrafish embryos and
larval eyes
For extraction of retinoids and carotenoids, freshly fertilized eggs were
collected. For determination of the retinoid content the eggs were homogenized
in 500 µl 2 M NH2OH.
HPLC system 1
Extraction of retinoids and HPLC analysis were carried out as previously
described (von Lintig and Vogt,
2000). For quantification of the molar amounts, peak integrals
were scaled with defined amounts of reference substances. The reference
substances all-trans and 13-cis retinol and retinal were purchased (Sigma,
Germany); 11-cis retinal was isolated from dark adapted bovine eyes.
Peak integrals were calculated using the 32 Karat software (Beckman
Instruments). For determining the retinoid content of larval eyes, the eyes of
dark-adapted larvae were removed by hand dissection with a scalpel under red
safety light. Extraction of retinoids and HPLC analyses were carried out as
previously described (von Lintig and Vogt,
2000
).
HPLC system 2
For determining the carotenoid content, the eggs were homogenized in 33%
ethanol PBS buffer (137 mM NaCl, 2.7 mM KCl, 7.3 mM
Na2HPO4, 1.47 mM KH2PO4, pH 7.2)
with a loose-fitting glass potter. Then 750 µl ethyl acetate/methyl acetate
(8:1 v/v) was added and vigorously vortexed. After centrifugation at 5000
g, the upper phase was collected. The extraction was repeated
twice and the collected organic phases were dried under a stream of nitrogen
and dissolved in HPLC solvent. Carotenoid analyses were performed on a
Hypersil 3 µm column (Knauer, Germany) on a Beckman System Gold (Beckman
Instruments) equipped with a multidiode array model 166 (Beckman Instruments).
The HPLC solvent was n-hexane/ethyl acetate (81:19, v/v) containing 15 µl
acetic acid per 100 ml solvent at a flow of 1 ml minute1.
For quantification of the molar amounts, peak integrals were scaled with
defined amounts of reference substances. The reference substances
ß-carotene, zeaxanthin and lutein were from Sigma; canthaxanthin and
cryptoxanthin were from Roth (Germany). Echinenone was obtained from Roche
(Switzerland). 4-Hydroxy-echinenone and isocryptoxanthin were obtained
respectively by reduction of canthaxanthin or echinenone with
NaBH4. Peak integrals were calculated using the 32 Karat software
(Beckman Instruments).
Photography
Live embryos and larvae where photographed in 2.5% methylcellulose/0.02%
3-aminobenzoic acid methylester (Sigma, Germany), stained whole-mount embryos
in 100% glycerol on a dissecting microscope (Leica MZ FLIII) with an Axiocam
(Zeiss). To assign tissue specificity of the bcox staining, the
embryos were embedded in Technovit 7100 (Kulzer Histotechnik, Germany) and
sectioned. Longitudinal and transversal cross sections of these preparations
were analyzed by microscopy.
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RESULTS |
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Provitamin A carotenoids exist in the zebrafish egg
The egg yolk of lower vertebrates, such as amphibians and teleosts,
generally contains large amounts of maternally derived vitamin A in the form
of retinal bound via a labile Schiff-base linkage to vitellogenins
(Plack et al., 1959;
Seki et al., 1987
;
Costaridis et al., 1996
;
Irie and Seki, 2002
). Until
now, it has been assumed that these retinoid stores are exclusively used for
embryonic retinoid metabolism (Costaridis
et al., 1996
). However, our results indicate that provitamin A
cleavage to retinal may impact, in addition, embryonic retinoid metabolism.
The prerequisite for this is the existence of provitamin A in the yolk, which
has so far not been demonstrated in zebrafish. Therefore, we extracted
lipophilic compounds from freshly fertilized eggs and subjected them to
HPLC-analyses (Fig. 3A,B). In
the egg extracts, we detected and identified various carotenoids by this
analysis. As summarized in Fig.
3, the major carotenoid was ß-carotene, but, additionally,
significant amounts of C4-substituted carotenoids (echinenone,
isocryptoxanthin, canthaxanthin, 4-hydroxy-echinenone) and
C3-substituted carotenoids (lutein, zeaxanthin) were present, as
judged by their retention times and spectral characteristics compared to
authentic standards. As quantified by HPLC analyses, yolk retinal existed in
substantial molar excess compared to carotenoids in a ratio of 100 to 6.
Costaridis et al. (Costaridis et al.,
1996
) have already shown that embryonic RA levels are low compared
with total egg yolk retinoid contents. To address the fate of these large
amounts of yolk retinal, we analyzed the retinoid content of the larval eyes
(4 dpf). Here, we recovered already
80% of the total egg retinal as
retinylesters and 11-cis-retinal with only traces of free retinol. Thus, the
major destination of the huge amounts of yolk retinal is the visual system.
Based on these analyses, we concluded that the yolk provitamin A content, even
though low compared with retinal content, would be sufficient to contribute to
embryonic RA signaling.
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Targeted gene knockdown of bcox interferes with hindbrain
development
As evidenced in mouse, chicken and zebrafish embryos, RA signaling is
crucial in patterning of caudal hindbrain and determining rhombomere identity
(Maden et al., 1996;
White et al., 2000
;
Niederreither et al., 1999
;
Niederreither et al., 2000
;
Dupé and Lumsden,
2001
). bcox mRNA expression was found in mesendoderm and
ventral mesoderm, suggesting that it may, in combination with raldh2,
contribute to generating RA during segmentation stages. To distinguish
individual hindbrain rhombomeres, we analyzed the expression of krx20
in r3 and r5 and the Eph-related receptor tyrosine kinase efna4 in
r1, r3 and r5. We found that r1 to r5 had a normal positional identity in
bcox morphants (Fig.
5A-D). We then investigated whether bcox morphants
develop patterning defects in hindbrain rhombomeres posterior to r5, as has
been described for zebrafish embryos mutant for neckless
(nls/raldh2) or no-fin (nof/raldh2), which are both
alleles of raldh2 (Begemann et
al., 2001
; Grandel et al.,
2002
). For this purpose, we analyzed valentino
(val) expression in r5 and r6, as well as expression of
hoxb4a, which is expressed with an anterior boundary between r6 and
r7. val appears to be normally expressed in r5 and r6 in
bcox morphants (Fig.
5E,F). We also performed whole-mount in situ hybridization for
combinations of krx20 and myod, as well as fgf8 and
val, to analyze whether the caudal hindbrain was extended and
posteriorized towards the somite boundary, as in zebrafish raldh2
mutants. bcox morphants did not develop these features of
no-fin and neckless mutants
(Fig. 5G-J).
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In summary, these analyses revealed that targeted knockdown of
bcox did not interfere with RA-dependent specification of rhombomere
identity during anteroposterior patterning of the neural tube. However,
reduced hoxb4a expression as well as altered ap2
expression in the hindbrain suggested that a lack of bcox activity
may interfere with later aspects of hindbrain development.
Impaired development of neural crest derivatives in the pharyngeal
region of bcox morphants
For a more detailed analysis of the craniofacial and branchial arch
phenotype, we stained the cartilage of the head skeleton with Alcian Blue. The
first and second arches were present in the morphants but malformed and less
chondrofied (Fig. 6A-D). The
first arch was smaller and pinched in the middle and its anterior portions
(Meckel's cartilages) were foreshortened and flattened in the front, while the
palatoquadrates were shortened as well as flared posteriorly. As seen in
lateral views, Meckel's cartilages projected ventrally. The hyoid arch was
completely distorted and the ceratohyales projected posteriorly rather than
anteriorly. From arches 3 to 7, the ceratobranchials were highly reduced or
absent in all morphants. In contrast to elements of the arch skeleton, the
neurocranium was properly formed in all bcox morphants analyzed
(n=61). Alcian Blue staining also revealed that pectoral fins were
present in bcox morphants but they were reduced in size and projected
laterally rather than caudally (Fig.
6F,G).
As shown above, bcox expression was found in cranial neural crest.
A direct role of RA signaling for neural crest cell differentiation and
survival in the pharyngeal region has been revealed by the phenotypes of
rara/rarb double mutants, as well as by raldh2
mutants (Dupé et al.,
1999; Begemann et al.,
2001
; Niederreither et al.,
1999
; Maden et al.,
1996
). We analyzed ap2
and dlx2
expression to visualize cranial neural crest contributing to branchial arches
in bcox morphants. At 24 hpf, ap2
expression was
reduced in cranial neural crest in the pharyngeal region as compared with
controls (Fig. 6H,I). At 31
hpf, dlx2 mRNA expression was detected in all streams of hindbrain
neural crest in both wild-type controls and bcox-MO-injected embryos,
but it was highly reduced in the posterior arch primordia of bcox
morphants (Fig. 6J,K). At 2
dpf, dlx2 mRNA expression was detectable in both bcox and
control morphants in mandibular and hyoid arch primordia, but its expression
was mostly absent in the posterior arch primordia of bcox-MO-treated
embryos (Fig. 6L,M). These
alterations in dlx2 expression indicated that targeted knockdown of
Bcox interferes with the development of posterior cranial neural crest streams
similar to what has also been reported for zebrafish raldh2 mutants
(Begemann et al., 2001
; Grandel
et al., 2001).
To pursue the further fate of pre-otic neural crest, we analyzed
dlx2 expression in 3 dpf larvae
(Fig. 6N,O). In controls,
dlx2 mRNA expression was downregulated, consistent with the beginning
of condensation and cartilage formation
(Ellies et al., 1997). By
contrast, in bcox morphants strong dlx2 expression persisted
in the pharyngeal region. To test whether this prolonged dlx2 mRNA
expression may be caused by a lack of RA, we applied exogenous RA at 2 dpf and
analyzed dlx2 expression 24 hours later. RA treatment resulted in a
downregulation of dlx2 mRNA expression (n=20), whereas in
DMSO-treated bcox morphants (n=20) dlx2 mRNA
persisted in the pharyngeal region (Fig.
6N-Q).
To summarize, bcox morphants develop a severe branchial arch phenotype with altered morphology of the mandible and hyoid arches, as well as a loss of gill arches. dlx2 expression was significantly altered in the neural crest, reflecting impairments in neural crest survival or differentiation particularly in the posterior arch primordia.
bcox is needed to establish the ventral retina
RA signaling has been shown to be crucial to establish the ventral part of
the retina during early eye development in zebrafish
(Marsh-Armstrong et al., 1994;
Hyatt et al., 1996
). We first
investigated the eyes of bcox morphants using Nomarski optics. At 24
hpf, the borders of the presumptive ventral retina were less distinct and the
choroid fissure was enlarged when compared with wild-type controls (data not
shown). Therefore, we analyzed the expression of pax6.1, pax2.1 and
shh: pax6.1 is expressed throughout the optic vesicles in
all cells of the prospective retina, the retinal pigment epithelium and lens
epithelium, whereas pax2.1 expression is restricted to the optic
stalk and retinal cells around the choroid fissure. These Pax gene expression
territories are influenced by midline signaling via Shh
(Macdonald et al., 1995
).
At 30 hpf, analyses of pax6.1 expression showed that the prospective retina was smaller and particularly reduced on the ventral side of the eyes (Fig. 7A,B,D,E), whereas the pax2.1 expression domain was extended and governed the enlarged choroid fissure of bcox morphants (Fig. 7G,H). Analyses of shh expression revealed that it was not significantly altered in the forebrain of bcox morphants at early (14 hpf, Fig. 7I,J) or later embryonic stages (24 hpf, data not shown). We then tried to phenocopy the MO-provoked impairments by applying the Raldh inhibitor citral to the incubation medium. Similar to bcox-MO treatments, citral treatments of embryos resulted in a reduction of the prospective ventral retina that was mirrored by a diminished pax6.1 expression domain (Fig. 7C,F).
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DISCUSSION |
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Both yolk-derived retinal and provitamin A are needed for hindbrain
development
Raldh2 is already expressed during gastrulation and somitogenesis in mouse,
chicken, Xenopus and zebrafish embryos. In several studies, it has
been conclusively shown that Raldh2-dependent conversion of retinal to RA is
required for anterioposterior patterning in the neural tube (for a review, see
Gavalas, 2002). bcox
is expressed in ventral cell layers adjacent to the yolk during segmentation,
raising the possibility that it may contribute retinal and act in coordination
with Raldh2 in RA-dependent hindbrain patterning. bcox morphants
showed normal position and identity of hindbrain rhombomeres. This clearly
excludes an early role of provitamin A conversion in RA-dependent
anterioposterior patterning of the neural tube. By treatment with the RAR
receptor pan-antagonist BMS493, Grandel et al. have shown that RA is required
as early as the 30% epiboly stage for patterning of the hindbrain and spinal
cord, as well as for the formation of pectoral fin buds in zebrafish
(Grandel et al., 2002
). In our
analyses, bcox expression was not detectable prior to segmentation
stages, thus strongly indicating that yolk-derived retinal is used for RA
generation by Raldh2.
However, the phenotype of bcox morphants suggests a developmental
role of provitamin A conversion during segmentation stages. Blocking RA
signaling using BMS493 beginning at tail bud stage also failed to interfere
with the patterning of hindbrain rhombomeres, but caused a diminished
hoxb4a expression at the caudal hindbrain boundary and malformed
pectoral fins (Grandel et al.,
2002), impairments like those observed in bcox morphants.
In addition, altered ap2
expression in the hindbrain in
bcox morphants is also observed in the zebrafish raldh2
mutant neckless (J.H., A. Barrallo, A. K. Ettl, E. Knapik and W.D.,
unpublished) or in our study of wild-type embryos treated with citral. This
RA-dependent induction of ap2
expression in the caudal
hindbrain is most probably required for the development of noradrenergic
neurons in medulla oblongata and area postrema (J.H., A. Barrallo, A. K. Ettl,
E. Knapik and W.D., unpublished). Thus, our data point to a biphasic
contribution of different sources for retinal during zebrafish hindbrain
development. At pre-segmentation stages, RA generation depends on preformed
yolk retinal, which must be liberated from its vitellogenin-bound form in
order to freely diffuse to raldh2-expressing cells. At segmentation
stages, the embryo relies at least in part on provitamin A and bcox
function to provide precursors for RA generation. This need for ongoing
provitamin A conversion to retinal for RA-dependent developmental processes is
strongly evident from the phenotypes of bcox morphants affecting the
eye, late differentiation in the hindbrain, as well as the development of
branchial arches. To exert this role during embryonic development, yolk
provitamin A first must be delivered to the site of action to become
metabolically converted to retinal by bcox function. Recently, we
identified a cell surface receptor (ninaD) that mediated the cellular
uptake of carotenoids in Drosophila
(Kiefer et al., 2002
).
Orthologs of ninaD also exist in vertebrates and may be involved in
provitamin A metabolism in vertebrates as well.
Patterning defects in pharyngeal arches
bcox morphants develop defects in neural crest derivatives, such
as diminished pigmentation and a malformed craniofacial skeleton. Our data
indicate that bcox itself is expressed in cranial and trunk neural
crest. So far, carotenoids and neural crest have been discussed solely with
respect to neural crest-derived pigmentation
(Kimler and Taylor, 2002). Our
analyses suggest an additional role of carotenoids as precursors for retinoids
for subsequent RA generation and signaling in neural crest contributing to the
craniofacial skeleton. Several studies already showed that RA is indispensable
for the development of caudal branchial arches in mouse, chicken and zebrafish
(Niederreither et al., 1999
;
Maden et al., 1996
;
Wendling et al., 2000
;
Begemann et al., 2001
). As
discussed above, hindbrain segmentation was not altered in bcox
morphants, thus excluding that defects in neural crest cell migration and
branchial arches may be secondary to defects in anterioposterior hindbrain
patterning. Emigration of all streams of hindbrain neural crest was also
verified by the analyses of dlx2 expression, showing that targeted
knockdown of bcox may interfere with neural crest cells at later
stages, affecting differentiation and potentially cell survival. Consistently,
dlx2 staining in posterior arch primordia was highly reduced already
at 31 hpf and mostly abolished at 2 dpf in bcox morphants. Similar
impairments have been reported in zebrafish raldh2 mutants,
indicating that bcox and raldh2 may act sequentially in RA
generation in caudal arch primordia
(Begemann et al., 2001
;
Grandel et al., 2002
).
Interestingly, raldh2 is expressed in the caudal part of branchial
arch primordia but not in neural crest
(Grandel et al., 2002
).
Therefore, retinoids synthesized via Bcox in neural crest cells may be
delivered to raldh2-expressing cells. For example, a close
association of migrating neural crest cells and raldh2-expressing
mesodermal cells has been shown in chick embryos
(Berggren et al., 1999
). This
bcox-dependent RA signal may then induce differentiation and prevent
apoptosis in mesoderm/endoderm or neural crest or in all tissues. The
transport of retinal, the initial product of ß-carotene cleavage, between
bcox and raldh2-expressing cells may additionally involve
retinoid-oxidizing as well as retinoid-binding proteins. Even though so far we
cannot exclude the expression of a different type of raldh in
zebrafish neural crest, the loss of gill arches in bcox morphants and
raldh2 mutants supports a signaling mechanism in which bcox
may locally supply retinal.
The altered morphology of the mandible and hyoid arch in bcox
morphants indicate a putative role of bcox for the development of
anterior arches. Here, in contrast to gill arches, dlx2 expression
was not abolished, but rather increased and its expression persisted at
developmental time points when it was downregulated in controls at the
beginning of condensation and cartilage formation
(Ellies et al., 1997). This
excludes a need of RA for the survival of pre-otic neural crest but, rather
suggesting a role of bcox for the differentiation of these cells at
later embryonic stages. The prolonged dlx2 expression in anterior
neural crest streams was downregulated by applying exogenous RA, thus
substantiating that targeted knockdown of bcox interferes with
RA-signaling here. As a result, the mandible and hyoid arch possess an altered
morphology and are less chondrofied in bcox morphants than in
controls. Malformations in first arch derived skeletal structures have been
also reported for Rara/Rarg double null mice
(Lohnes et al., 1994
). In
addition, effects of RA on chondrogenesis and synthesis of extra-cellular
matrix macromolecules have been shown, i.e. in chicken craniofacial mesenchyme
(Sakai and Langille, 1992
). As
raldh2 is not expressed in anterior parts of the developing pharynx
(Grandel et al., 2002
), it
must be supposed that a different type of raldh may act downstream of
bcox in the pharyngeal region at later developmental time points.
Besides branchial arches, neural crest-derived pigmentation was also impaired
in bcox morphants, indicating that bcox may play a more
general role in the development of cranial and trunk neural crest
derivatives.
Impairments of the development of the ventral retina in bcox
morphants
The eyes are known to be especially vulnerable against imbalances in
retinoid levels during their development. Older studies already revealed that
vitamin A deprivation of mammals during distinct developmental time windows
results in microphthalmia of otherwise normal offspring
(Warkany and Schraffenberger,
1946). Molecular analyses suggest a role of RA in patterning the
dorsal-ventral axis of the eyes, as Rara and Rarb double
mutant mice develop a reduced ventral retina and, as a consequence,
microphthalmia (Kastner et al.,
1994
). A role of RA in specifying ventral characteristics of the
retina has been also shown in zebrafish. Exogenous RA application results in
proliferation of cells in the ventral region of the retina, leading to retina
duplication (Hyatt et al.,
1992
; Hyatt et al.,
1996
), whereas inhibition of endogenous embryonic RA synthesis by
citral results, by contrast, in strong reduction of the ventral retina
(Marsh-Armstrong et al.,
1994
). A crucial process with respect to RA perturbations is the
formation of the optic primordia (Hyatt et
al., 1992
). bcox is expressed at the ventral side of the
primordia, beginning with early somite stages. bcox knockdown
resulted in smaller eyes and on the cellular level in shortened and less
stratified outer segments of the photoreceptors at larval stages. At 30 hpf,
bcox morphants already exhibited morphological changes, i.e. a
reduced ventral retina and an enlarged choroid fissure. At the molecular
level, this was mirrored by reduced pax6.1 expression in the
presumptive retina. Comparable morphological changes and reduced
pax6.1 expression were also provoked by treatments with the Raldh
inhibitor citral (this study)
(Marsh-Armstrong et al.,
1994
), thus showing that targeted knockdown of bcox
function phenocopies RA deficiency during eye development. A reciprocal
regulation of pax6 and pax2 expression territories in
establishing the boundary between the optic cup and stalk has been previously
shown in mice (Schwarz et al.,
2000
). The analysis of pax2.1 expression revealed an
enlarged expression domain in the eyes of bcox morphants. However,
pax2.1 expression remained restricted to the ventral part of the eyes
(choroid fissure and optic stalk) and did not extend dorsally into the
prospective neural retina, as has been described in zebrafish exposed to
exogenous RA (Hyatt et al.,
1996
). This indicates that the expanded pax2.1 expression
domain is most probably a consequence of the loss of pax6.1
expression in the ventral retina, rather than of a per se upregulated
pax2.1 expression. In addition, shh expression is normal,
indicating no alterations in midline signaling in bcox morphants. A
role for RA signaling in maintaining shh expression during forebrain
development has recently been suggested for chicken embryos
(Schneider et al., 2001
).
Zymographic assays revealed that RA is first synthesized in the ventral
retina beginning at the 12-somite stage in zebrafish, and that two different
types of Raldh exist in the eyes
(Marsh-Armstrong et al.,
1994). In transgenic reporter zebrafish, a RA-responsive reporter
gene also responds to RA first in the ventral retina and RA production in the
dorsal retina occurs only later
(Perz-Edwards et al., 2001
).
In zebrafish, raldh2 expression is restricted to the dorsal retina
(Grandel et al., 2002
). In
mice, a new type of Raldh, Raldh3, has been identified. It is
expressed in the ventral retina during eye development and is most probably
responsible for RA generation here (Mic et
al., 2000
; Li et al.,
2000
). The fact that zebrafish raldh2 mutants show no
obvious eye phenotype (Begemann et al.,
2001
; Grandel et al.,
2002
) indicates that a Raldh3 orthologue might also exist
in zebrafish that could act downstream of Bcox. Based on our data, loss of
this putative Raldh3 function might result in an eye phenotype
comparable with the one described here for bcox morphants.
Conclusions
Our study reveals that bcox and provitamin A carotenoids play a
vital role during zebrafish development. Comparable impairments as in
bcox morphants have also been described in zebrafish raldh2
mutants or were provoked by citral treatments of wild-type embryos
(Begemann et al., 2001; Grandel
et al., 2001; Marsh-Armstrong et al.,
1994
). Thus, our analyses identify the
ß,ß-carotene-15,15'-oxygenase acting upstream of Raldhs and
strongly suggest that provitamin A conversion is the prerequisite for
RA-signaling in several distinct developmental processes in zebrafish embryos.
The use of the non-toxic provitamin instead of preformed yolk retinal for
RA-signaling processes may provide an additional control mechanism to finely
balance retinoid levels at the cellular level in local tissue environments. We
cannot yet unequivocally exclude the possibility that any of the ADH or SDR
family members acts in between provitamin A conversion and RA generation. In
zebrafish, application of the ADH1 inhibitor 4-methylprazole did not interfere
with development (Costaridis et al.,
1996
). Additionally, Adh1- and Adh4-null mice
show no impairments during development when sufficient vitamin A is available
for the embryo (Deltour et al.,
1997
; Deltour et al.,
1999
). Recent results indicate that Adh3, a ubiquitously
expressed retinol-oxidizing enzyme, is needed for mouse development
(Molotkov et al., 2002
).
Interestingly, embryonic expression of a bcox homologue has been also
reported in mice, indicating that a developmental function of provitamin A
conversion may exist in higher vertebrates as well
(Redmond et al., 2001
). Thus,
Adh3 and bcox may both play a role during embryonic
development by delivering retinal for certain aspects of retinoid signaling.
Our findings in zebrafish promise to elucidate new aspects for provitamin A
and carotene-oxygenases in vertebrate development.
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ACKNOWLEDGMENTS |
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