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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02706
1 HHMI and Division of Basic Science, Fred Hutchinson Cancer Research Center,
1100 Fairview Ave. N., Seattle, WA 98109-1024, USA.
2 Medical Scientist Training Program and Molecular and Cellular Biology Program,
University of Washington, Seattle, WA 98195-7470, USA.
Author for correspondence (e-mail:
cmoens{at}fhcrc.org)
Accepted 18 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Retinoic acid, Hindbrain, Cyp26, Hox, Morphogen, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Dupe and Lumsden,
2001; Maden et al.,
1996; Niederreither et al.,
1999). RA is produced in the anterior paraxial mesoderm by the
activity of aldehyde dehydrogenase 1 family, member A2 (Aldh1a2), which
oxidizes retinal to RA (Begemann et al.,
2001; Gavalas,
2002; Niederreither et al.,
1999). RA either diffuses or is transported from the paraxial
mesoderm into the adjacent central nervous system. RA directly regulates gene
expression through its nuclear hormone receptor (RAR) and co-receptor (RXR),
which bind RA response elements (RAREs) in the enhancers of target genes
(Bastien and Rochette-Egly,
2004). In the hindbrain, RA regulates the expression of
3'-Hox genes through direct (in the case of Hox-1 and Hox-4 genes) or
indirect (in the case of Hox-3 genes) mechanisms
(Gould et al., 1998;
Hernandez et al., 2004;
Marshall et al., 1994;
Nolte et al., 2003;
Studer et al., 1994;
Zhang et al., 2000). Other
anterior RAresponsive genes (Hox-1 family genes) are expressed earlier and at
lower RA concentrations than the more-posterior RA-responsive genes (Hox-4
family genes) (Dupe and Lumsden,
2001; Maves and Kimmel,
2005; Simeone et al.,
1990). Based on the effects of switching the RAREs of the Hox-1
and Hox-4 genes, Gould et al. proposed that Hox-1 genes are expressed at more
anterior levels than Hox-4 genes because their RAREs are more sensitive to RA
(Gould et al., 1998).
These data have lead to a model in which a continuous spatio-temporal
gradient of RA through the hindbrain generates nested domains of RA-responsive
gene expression. These domains are then resolved by secondary mechanisms into
non-overlapping domains that correspond with the morphological segments of the
hindbrain - the rhombomeres (r). However, a number of observations suggest
that an RA gradient is neither detectable nor required for normal hindbrain
development. First and foremost, embryos depleted of endogenous RA can be
fully rescued by a uniform concentration of exogenous RA
(Begemann et al., 2004;
Begemann et al., 2001;
Gale et al., 1999;
Grandel et al., 2002;
Mic et al., 2002;
Niederreither et al., 2000).
Second, this rescue can be accomplished by a range of RA concentrations and
over a range of developmental stages (Dupe
and Lumsden, 2001; Maves and
Kimmel, 2005). Third, when RA responsiveness is measured by the
expression of a RARE-LacZ reporter, no gradient of expression is detected in
the hindbrain. Instead, distinct boundaries of reporter expression that shift
over time are detected (Rossant et al.,
1991; Sirbu et al.,
2005). Finally, in contrast to earlier findings
(Gould et al., 1998), recent
evidence has suggested that, in the context of their intact enhancers, a Hox-1
RARE is equally responsive to RA as a Hox-4 RARE
(Nolte et al., 2003).
These data suggest that cells in the presumptive hindbrain neuroepithelium
can be patterned by RA in a manner that is independent both of concentration
and of duration of exposure, necessitating a new model for RA-dependent
hindbrain patterning. Here, we propose such a model based on the hindbrain
patterning defects caused by the prevention of RA metabolism by the cytochrome
P450 enzymes of the Cyp26 class. The Cyp26 enzymes (Cyp26a1, Cyp26b1 and
Cyp26c1) have been proposed to function in the regulation of RA-dependent gene
expression through their ability to metabolize RA into hydroxylated polar
derivatives (Fujii et al.,
1997; White et al.,
1996). In the mouse tailbud and limbs, loss of Cyp26 function
leads to increased RA-dependent gene expression, spina bifida and caudal
agenesis similar to the teratogenic effects of high concentrations of
exogenous RA (Abu-Abed et al.,
2001; Sakai et al.,
2001; Yashiro et al.,
2004).
With regard to hindbrain patterning, cyp26a1 is expressed during
gastrulation in the anterior neurectoderm
(Dobbs-McAuliffe et al., 2004;
Kudoh et al., 2002). Based on
this expression domain, it was hypothesized that localized regions of RA
synthesis in the anterior trunk mesoderm and degradation in the anterior
neural plate provide a classical `source-and-sink' mechanism for the spatial
regulation of RA in the central nervous system
(Kudoh et al., 2002;
Swindell et al., 1999).
However, cyp26a1 mutants in fish and mouse exhibit relatively subtle
hindbrain-patterning defects inconsistent with a global role for
cyp26a1 in hindbrain patterning
(Abu-Abed et al., 2001;
Emoto et al., 2005;
Kudoh et al., 2002;
Sakai et al., 2001). The
recent identification of other cyp26 genes has suggested that these may
participate in shaping RA responsiveness in the hindbrain
(Abu-Abed et al., 2002;
Gu et al., 2005;
MacLean et al., 2001;
Reijntjes et al., 2005;
Reijntjes et al., 2004;
Sirbu et al., 2005;
Tahayato et al., 2003;
Taimi et al., 2004;
Zhao et al., 2005). Here, we
demonstrate that the zebrafish orthologs of mammalian CYP26B1 and CYP26C1
function redundantly with cyp26a1 to pattern the hindbrain, because
embryos depleted of all three proteins exhibit a profound posterior
transformation of the hindbrain. Furthermore, we demonstrate that cyp26 genes
are responsible for the ability of exogenous RA to rescue embryos depleted of
endogenous sources of RA. In embryos depleted of Cyp26 activity, the low RA
concentrations that normally rescue the RA-depleted hindbrain are highly
teratogenic. Based on our results, we present a `gradient-free' model for
RA-dependent hindbrain patterning in which the spatially regulated
inactivation of RA by Cyp26 enzymes is responsible for the establishment of
RA-responsive gene-expression domains in the hindbrain.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and we cloned
the 5' end of the coding sequence with the SMART RACE kit (Clontech).
cyp26c1 was identified in a Blast search of the zebrafish genome
sequence using the human CYP26C1 protein sequence and was then amplified from
12 hour post fertilization (hpf) whole-zebrafish-embryo cDNA.
Morpholinos, RNA in situ hybridizations and genotyping
Table 1 summarizes the
sequences of the morpholinos (MOs) used in this study, the combinatorial
depletion experiments performed and their outcomes. To determine the efficacy
of cyp26b1 MO1, we made a cyp26b1-GFP fusion construct by
cloning a fragment of the 5' UTR and 5' coding sequence including
the MO target sequence into pCS2-GFPLTXLT. The resulting plasmid
was linearized with NotI and mRNA was prepared for injection with the
SP6 mMessage mMachine kit (Ambion). Embryos were injected with 470 pg mRNA
plus 5 ng MO. Embryo lysates were prepared as described
(Waskiewicz et al., 2001),
separated on 4-12% NuPAGE Bis-Tris gels and transferred to Invitrolon PVDF
membranes (Invitrogen). For immunoblotting, we used anti-GFP (Torrey Pines,
1:2000) and anti-Actin (SantaCruz, sc1616, 1:200), and proteins were detected
with SuperSignal West Dura substrate (Pierce) according to the manufacturer's
directions. Quantitation of the GFP bands showed that cyp26b1 MO
blocked 95-98% of the translation of the injected cyp26b1-GFP
mRNA.
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All of the experiments described in this manuscript used cyp26b1
MO1 and cyp26c1 MO1; however, cyp26b1 MO2 and
cyp26c1 MO2 gave the same phenotypes. Unlike cyp26c1 MO1,
cyp26c1 MO2 was toxic at higher concentrations. Our control MO was
targeted to the dead-end mRNA and eliminates primordial germ cells
but does not affect other aspects of development
(Weidinger et al., 2003). To
assay for redundancy between the cyp26 genes, MOs were injected alone or in
combination into embryos from a cyp26a1+/- intercross
(Emoto et al., 2005). In order
to control for nonspecific effects due to MO injections, all embryos were
injected with a total of 5 ng MO as determined by measuring the diameter of
the injected bolus in mineral oil (see
Table 1).
Two-color RNA in situ hybridizations were performed, essentially as
described (Prince et al.,
1998), except that Iodo-Nitrotetrazolium Violet (Sigma) was used
as the red Alkaline Phosphatase substrate. Embryos were de-yolked and
flat-mounted for photomicroscopy using a Zeiss Axioplan II microscope. After
photographing, individual embryos were un-mounted and genotyped for the
cyp26a1 mutation as described
(Emoto et al., 2005).
Drug treatments
Dechorionated embryos from wild-type or cyp26a1+/-
parents were incubated in the dark in pharmacological agonists and antagonists
of the RAmetabolism pathway as follows: 4-(diethylamino)benzaldehyde [DEAB, an
inhibitor of retinaldehyde dehydrogenases
(Russo et al., 1988);
Aldrich]: 10 µM, beginning at 50% epiboly [5.25 hpf
(Kimmel et al., 1995)];
R115866, a specific inhibitor of Cyp26 enzymes (Janssen Pharmaceutica): 10
µM, beginning at dome stage (4.33 hpf); all-trans RA (Sigma): 0.1-100 nM,
beginning at 50% epiboly.
In each case, the compound was diluted to 1000 times its final concentration in DMSO and then diluted 1000-fold in embryo medium. Controls were treated with carrier alone (0.1% DMSO). For retinal treatments, embryos were injected at the one-cell stage with 1 nl of 20 pmol/nl all-trans retinal in DMSO (Sigma), and controls were injected with 1 nl DMSO alone.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We cloned zebrafish homologs of cyp26b1 and
cyp26c1, and examined their expression during development.
Cyp26c1 has been previously described as cyp26d1
(Gu et al., 2005) and
cyp26b1-like (Kawakami et al.,
2005) (ZDB gene 050714-2). Based on two lines of evidence, we
argue that this gene is in fact the ortholog of mammalian CYP26C1.
First, phylogenetic analysis of zebrafish cyp26 genes places it in the same
clade as mouse and human CYP26C1 genes with moderate bootstrap
support (see Fig. S1A in the supplementary material). Second, CYP26A1
and CYP26C1 are adjacent to one another on human chromosome
10q23-q24, and current zebrafish genomic sequence data and radiation-hybrid
data places zebrafish cyp26a1 and cyp26c1, cyp26d1- and
cyp26b1-like in regions of zebrafish linkage groups (LGs) 12 and 17,
respectively, which show synteny to human chromosome 10 (see Fig. S1B in the
supplementary material) (Woods et al.,
2005). Because draft genomes for other tetrapod vertebrates also
show CYP26A1 and CYP26C1 as adjacent genes, it is likely
that they were adjacent genes in the ancestral vertebrate genome and that,
following the proposed genome duplication early in teleost evolution, the
duplicate of cyp26c1 was lost from LG 12 and the duplicate of
cyp26a1 was lost from LG 17. Henceforth, we refer to the gene
previously named cyp26d1 or cyp26b1-like as
cyp26c1.
The expression patterns of zebrafish cyp26a1, cyp26b1 and
cyp26c1 have been described
(Dobbs-McAuliffe et al., 2004;
Gu et al., 2005;
Kudoh et al., 2002;
Zhao et al., 2005). We focus
here on their expression during hindbrain development. During gastrulation,
cyp26a1 is expressed in the anterior neurectoderm (bracket in
Fig. 1B) and in a narrow domain
at the margin at 8.5 hpf (arrowhead in Fig.
1B). The posterior limit of cyp26a1 expression at 8.5 hpf
abuts the anterior limit of hoxb1b expression at the r3-r4 boundary
(Kudoh et al., 2002), but
rapidly recedes anteriorly to lie at the r2-r3 boundary at 10 hpf and lies
further anterior still at 11 hpf (Fig.
1F,G). As described previously, cyp26a1 is directly
RA-inducible, even at sub-teratogenic concentrations of RA (5 nM), which cause
the ectodermal domain of expression to expand towards the margin
(Fig. 1C)
(Dobbs-McAuliffe et al., 2004;
Kudoh et al., 2002;
Loudig et al., 2000;
White et al., 1996). In spite
of its strong RA-inducibility, the early anterior neurectodermal expression of
cyp26a1 is established independently of RA, evident because it is
unaffected in embryos treated with 4-(diethylamino)benzaldehyde (DEAB), a
specific inhibitor of retinaldehyde dehydrogenase
(Fig. 1D)
(Dobbs-McAuliffe et al., 2004;
Sirbu et al., 2005).
Throughout the hindbrain-patterning period, neurectodermal cyp26a1
expression lies significantly anterior to that of aldh1a2, which is
restricted to the anterior mesoderm (Fig.
1E,H,I) (Dobbs-McAuliffe et
al., 2004; Kudoh et al.,
2002; Sirbu et al.,
2005; Swindell et al.,
1999). cyp26a1 is also expressed in the tailbud and in a
crescent in the anterior trunk mesoderm immediately anterior to the
aldh1a2-expressing domain (brackets in
Fig. 1F,G).
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Unlike cyp26a1, neither cyp26b1 nor cyp26c1 are
globally upregulated by exogenous RA (data not shown, and see Fig. S3B,E in
the supplementary material). RA is also not required for the normal onset of
their expression, evident because both genes are expressed in DEAB-treated
embryos (see Fig. S3C,F in the supplementary material). However, we did
observe effects on the expression of both cyp26b1 and
cyp26c1 at the 3-somite stage that suggest that both genes are
affected indirectly by RA-dependent patterning events in the hindbrain (see
Fig. S3 in the supplementary material). Briefly, in 100 nM RA, r4 is expanded
anteriorly, and with it the r4 expression of cyp26b1 and
cyp26c1, whereas in 10 µM DEAB r2 and r3 are expanded posteriorly,
and with them the r2 and r3 expression of cyp26b1 and
cyp26c1. Sirbu et al. (Sirbu et
al., 2005) showed that cyp26c1 expression in r4 is
dependent upon RA. We do not see clear evidence of this in the zebrafish,
although the r4 domain of cyp26c1 expression is consistently reduced
at the 3-somite stage in DEAB-treated embryos (see Fig. S3B in the
supplementary material).
cyp26b1 and cyp26c1 contribute to normal hindbrain
patterning We tested the function of Cyp26b1 and Cyp26c1 by knocking-down
their function using antisense MOs. We performed all of our experiments in
embryos generated by intercrossing cyp26a1 heterozygotes so that we
could examine Cyp26b1 and Cyp26c1 function both in the presence and in the
absence of Cyp26a1 function (Table
1). The hindbrain phenotype of cyp26a1 mutants is subtle:
r4 [marked by hoxb1a and bounded by the r3 and r5 stripes of
krox20 (also known as egr2b - Zebrafish Information
Network)] is slightly expanded in length and the anterior hindbrain (r1-r3) is
slightly reduced (Fig. 2A,B,
Fig. 4A,B and see Fig. S4A,B in
the supplementary material) (Emoto et al.,
2005). Furthermore, the posterior-most hindbrain - marked by high
levels of hoxd4 expression, and comprising r7 and the long
unsegmented `vagal' rhombomere, r8, which lies between the segmented hindbrain
and the first somite (Lumsden,
1990) - is expanded in length as described previously
(Emoto et al., 2005)
(Fig. 2A,B and
Fig. 4A,B).
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Embryos depleted of all three Cyp26 proteins have a strongly posteriorized
hindbrain (Fig. 2G-L and see
Fig. S4G,H in the supplementary material). Both r3 and r5 are eliminated
(Fig. 2G,H) and r4 abuts the
cerebellum (Fig. 2I,J and see
Fig. S4G,H in the supplementary material). The anterior limit of
vhnf1 expression, which, by the 8-somite stage (13 hpf), marks the
r5-r6 boundary, is also shifted to abut the cerebellum (arrowheads in
Fig. 2K,L). The r6-r7 boundary
of hoxd4 expression is similarly shifted, coming to lie within a few
cell diameters of cerebellum (arrowhead in
Fig. 2H). Thus, three
RA-responsive genes (hoxb1a, vhnf1 and hoxd4) that normally
form nested expression domains in the hindbrain are all expanded into the
anterior-most hindbrain in embryos depleted of all three Cyp26 proteins. In
spite of this strong transformation of the hindbrain, the patterning of the
mid- and fore-brain, marked by pax2a, otx2, dlx2a and eomes,
appears unaffected, except for a decrease in length detectable in
cyp26a1 single mutants (data not shown)
(Emoto et al., 2005).
We examined the neuronal organization of Cyp26-depleted embryos. In cyp26a1+ embryos injected with control MOs or with cyp26b1 and/or cyp26c1 MOs, we observed normal patterns of cranial and spinal motor-nerve differentiation (Fig. 2M). However, in cyp26a1-/- embryos injected with both cyp26b1 MO and cyp26c1 MO, the vagal neurons (nX in Fig. 2M,N) characteristic of r8 are expanded to the mid-hindbrain boundary (large arrowhead in Fig. 2M,N) and spinal motor roots (arrows in Fig. 2M,N) extend from hindbrain levels into a disorganized branchial region (small arrowheads in Fig. 2M,N). This occurs in spite of the fact that there are no somites to innervate at this level (data not shown). These neuronal phenotypes are consistent with our analysis of marker gene expression, in which the RA-inducible hox gene characteristic of r7-8 (hoxd4) is expanded anteriorly throughout the hindbrain region.
A pharmacological inhibitor of Cyp26 activity phenocopies Cyp26 depletion
Pharmacological antagonists that inhibit RA metabolism have been developed
as tools for the treatment of dermatological diseases and cancer
(Njar, 2002;
Njar et al., 2006). The
compound R115866 is a highly selective antagonist of Cyp26a1 activity in
vitro, and exerts retinoidal effects in adult rats
(Stoppie et al., 2000). Its
effects on Cyp26b1 and Cyp26c1 have not been examined. We observed that
treating zebrafish embryos with 10 µM R115866 caused a phenotype identical
to that of embryos depleted of all three Cyp26 enzymes (compare
Fig. 2J,H with
Fig. 3B,D). This suggests that
R115866 inhibits Cyp26b1 and Cyp26c1 as effectively as does knocking-down
their expression with MOs, and confirms our above observation that Cyp26
activity is essential for normal hindbrain patterning. The effects of R115866
treatment are completely reversed by the addition of DEAB, so that embryos
treated with both drugs resemble embryos treated with DEAB alone
(Fig. 3E,F). This demonstrates
that, as for other phenotypes observed in cyp26a1-mutant fish and
mice (Emoto et al., 2005;
Niederreither et al., 2002),
the posteriorized hindbrain phenotype caused by blocking all Cyp26 activity is
due to the accumulation of excess RA and not to the absence of bioactive
Cyp26-generated RA derivatives. Although such derivatives have been observed
to have significant retinoidal effects in cells and in embryos, and have been
postulated to have functions in vivo
(Idres et al., 2002;
Pijnappel et al., 1993), we
see no evidence for their having a role in hindbrain patterning.
Cyp26a1 protects against RA teratogenicity.
The long-standing observation that depletion of endogenous RA can be
rescued by treatment with a low concentration of exogenous RA demonstrates
that a RA gradient is not strictly necessary for hindbrain patterning
(Begemann et al., 2004;
Begemann et al., 2001;
Gale et al., 1999;
Niederreither et al., 2000).
However, the basis of this rescue phenomenon has not been determined, and has
significant implications for the mechanism of hindbrain patterning. We
hypothesized that Cyp26 enzymes enable this rescue by inactivating exogenous
RA in a patterned manner. We tested the roles of the cyp26 genes by performing
the RA-rescue experiment in Cyp26-depleted embryos.
The effects of blocking RA synthesis with DEAB in zebrafish have been
described previously (Begemann et al.,
2004; Maves and Kimmel,
2005). They include the loss of posterior hindbrain identities
(r5-r8, Fig. 4C), expansion of
anterior hindbrain identities (r2-r4), and a dramatic anterior shift of
paraxial- and lateral-plate mesoderm-derived tissues (pronephros and somites;
Fig. 4C). In wild-type
(cyp26a1+/+ and cyp26a1+/-)
DEAB-treated embryos, this phenotype is rescued by treatment with between 0.5
and 10 nM RA: concentrations that are non-teratogenic or weakly teratogenic in
wild-type embryos (Fig.
4E,G,I,K and data not shown). In the experiments described below,
we used 5 nM RA as our `rescuing' concentration. Whereas in wild-type embryos
5 nM RA is non-teratogenic, it strongly posteriorizes
cyp26a1-/- embryos, either in the presence or in the
absence of DEAB, causing anterior expansion of r7-r8 identity
(Fig. 4E,F and data not shown)
and the loss of all brain regions anterior to r7: r3 and r5 (marked by
krox20; Fig.
4E,F,I-L); r4 (marked by hoxb1a;
Fig. 4G,H); the cerebellum and
posterior tectum (marked by en3;
Fig. 4G-J); the diencephalon
and midbrain (marked by otx2; Fig.
4K,L); and the telencephalon and eyes [marked by dlx2
(Fig. 4I,J) and by
eomes (data not shown)]. Embryos posteriorized in this manner
typically exhibited an accordion-like folding of the anterior neural tube.
Exactly the same effects are observed in cyp26a1-/-
embryos treated with 5 nM RA in the absence of DEAB (data not shown). This
phenotype strongly resembles the effects of 40-fold-higher levels of RA on
wild-type embryos (inset in Fig.
4J), demonstrating that it is the ability of cyp26a1 to
inactivate RA that enables RA-deficient embryos to be rescued by exogenous RA.
Furthermore, these results demonstrate that cyp26a1 is able to
protect embryos from the potentially teratogenic effects of low concentrations
of RA. We did not see a similar sensitivity to exogenous RA in
cyp26b1 and/or cyp26c1 MO-injected embryos.
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cyp26a1 protects against potentially teratogenic RA precursors
Our findings demonstrate that cyp26a1 protects against the
potentially teratogenic effects of RA. Maternally-derived RA is present at
very low levels in zebrafish eggs and early embryos prior to the onset of
embryonic RA synthesis, and is therefore unlikely to be a teratogenic risk
(Costaridis et al., 1996).
However, the levels of maternally loaded retinal - the immediate precursor of
RA - are higher (9 pmol/egg) (Costaridis et
al., 1996; Lampert et al.,
2003). We asked whether Cyp26a1 protects against teratogenicity of
RA precursors. We increased retinal levels in wild-type and
cyp26a1-/- eggs by injecting retinal directly into the
yolk of one-cell stage embryos. Although wild-type embryos tolerate over
ten-times the normal amount of retinal in the yolk (data not shown),
cyp26a1-/- embryos are strongly posteriorized by only a
three-times the normal amount of retinal (27 pmol;
Fig. 6). The teratogenic
effects of a small increase in RA precursor in the absence of Cyp26a1
demonstrates that Cyp26a1 may normally play an important role in protecting
the embryo against the potentially teratogenic effects of maternally derived
RA precursors. They also suggest that the RA biosynthetic enzyme Aldh1a2 is
unable to buffer changes in the levels of its substrate. Because retinal is
itself derived directly from dietary vitamin A, it may be expected to
fluctuate depending on maternal diet. These observations emphasize the crucial
importance of a tightly regulated RA-degradative pathway in nervous system
patterning.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We have identified a crucial role for Cyp26 RA-metabolizing enzymes in establishing hindbrain pattern, because depleting them alone and in combination leads to a progressive posteriorization of the hindbrain. In fully Cyp26-depleted embryos, three RA-dependent genes that normally form nested expression domains with distinct anterior limits (hoxb1a, r3-r4; vhnf1, r4-r5; and hoxd4, r6-r7) are all expanded up to the anterior-most hindbrain (Fig. 7A). Based on the dynamic expression of the cyp26 genes in the hindbrain and on the effects of depleting embryos of Cyp26 activity, we propose a gradient-free model for RA-dependent events in hindbrain patterning, in which RA degradation by Cyp26 enzymes determines progressively more-posterior limits of RA-dependent gene expression in a step-wise manner (Fig. 7B). We note that cyp26 genes are expressed similarly, although not identically, in tetrapods, predicting a similar combinatorial role for Cyp26 enzymes in mammalian hindbrain development.
In step 1, complete by 9 hpf, the anterior limit of hoxb1b and
hoxb1a - the functional homologs of mammalian HOXA1 and
HOXB1 - are established by the posterior limit of cyp26a1
expression. This event establishes the r3-r4 boundary
(Kudoh et al., 2002), the
first morphological boundary in the hindbrain
(Moens et al., 1998). This
function can be compensated for by cyp26c1, because the anterior
limit of r4 is strongly affected only in the absence of both cyp26a1
and cyp26c1. In step 2, complete by 11 hpf, the anterior limit of the
next RA-responsive gene, vhnf1, is determined by the posterior limit
of cyp26c1 expression at the r4-r5 boundary. This function can be
partially compensated for by cyp26b1. In step 3, complete by 12 hpf,
the anterior limit of the last RA-responsive gene, hoxd4, is
determined by the posterior limit of cyp26c1 at the r6-r7 boundary, a
function that can also be compensated for by the overlapping expression of
cyp26b1. Some of the mechanistic underpinnings of this model and its
broader implications are discussed further below.
A similar model was previously proposed based on the correspondence between
cyp26a1 and cyp26c1 expression domains and boundaries of
RA-dependent reporter-gene expression in the mouse
(Sirbu et al., 2005). These
authors predicted that cyp26a1 establishes the r2-r3 boundary and
that cyp26c1 subsequently establishes the r4-r5 boundary. Our
combinatorial functional analysis of cyp26 genes confirms this model
in the general sense that Cyp26 activity determines sequential boundaries of
RA-responsive gene expression in the hindbrain. However, our observations
demonstrate a different and broader role for cyp26 genes in hindbrain
patterning, involving all three cyp26 genes functioning to establish three
sequential RA-responsiveness boundaries: r3-r4, r4-r5 and r6-r7. We do not
observe a function for Cyp26 enzymes at the r2-r3 boundary: r1-r3 are entirely
lost while r4 identity shifts anteriorly to abut the forming cerebellum.
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; Kudoh et al.,
2002; Sirbu et al.,
2005), whereas both cyp26b1 and cyp26c1 mark,
sequentially, the r4-r5 and r6-r7 boundaries. However, it is important to
notice that cyp26a1 is also expressed in the anterior trunk mesoderm
near the RA source, where it probably functions to reduce global RA levels
(Emoto et al., 2005;
Niederreither et al., 2002).
We propose that the severe posteriorization of Cyp26-depleted embryos results
from the combined effects of depleting segment-restricted Cyp26 activity
within the hindbrain and increasing global RA levels due to the loss of
Cyp26a1 activity in the anterior trunk mesoderm
(Fig. 7B).
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a and RARb - are expressed throughout the hindbrain, but
RARb mRNA levels are higher in the presumptive r5 and r6 whereas
RARa mRNA levels are higher posterior to the presumptive r6-r7 boundary
(Hale et al., 2006).
Furthermore, RXR
, a RA co-receptor, is exclusively expressed posterior
to the r6-r7 boundary (Tallafuss et al.,
2006). By increasing the RA response, these nonhomogeneously
distributed RARs and RXRs may help to establish the r4-r5 and r6-r7
boundaries. A third possibility is that spatially restricted transcription
factors repress RA-responsive gene expression even when ligand and receptor
are present. Iro7 (also known as Irx7 - Zebrafish Information Network) is a
TALE homeodomain protein expressed in the anterior hindbrain that represses
vhnf1 expression anterior to the r4-r5 boundary
(Lecaudey et al., 2004). Other
TALE homeodomain proteins have been shown to repress transcription from
retinoid-responsive elements by binding to RXR retinoid receptors and
recruiting general corepressors to the complex
(Bartholin et al., 2006). Thus,
Iro7 may compensate for Cyp26b1 and Cyp26c1 by directly suppressing
RA-responsive gene expression anterior to the r4-r5 boundary. Finally, a
diffusion gradient of RA from its source in the anterior trunk mesoderm may
compensate for the absence of hindbrain Cyp26 expression. RA can act as a
classical morphogen, specifying distinct rhombomere identities at different
threshold concentrations (Dupe and
Lumsden, 2001; Maves and
Kimmel, 2005), and an RA gradient may initiate nested domains of
RA-responsive gene expression in the hindbrain when cyp26b1 and
cyp26c1 are depleted. We note that any or all of the mechanisms we
have proposed above (a diffusion gradient, receptor expression or other
transcription factors that modulate RAR activity) may contribute to hindbrain
patterning under normal circumstances. However, none of these mechanisms are
sufficient to limit RA responsiveness in the hindbrain when all three Cyp26
enzymes are depleted. cyp26a1 is required to establish hindbrain pattern in the absence of a localized source of RA We have shown that, in the zebrafish, cyp26a1 is essential for the ability of exogenous RA to rescue embryos in which endogenous RA synthesis is inhibited. Although 5 nM RA can fully rescue the hindbrain and anterior trunk patterning defects of wild-type embryos in which RA synthesis is inhibited with DEAB, in cyp26a1-/- embryos it causes a strong posteriorization similar to that normally caused by 40 times more RA. From this, we conclude that Cyp26a1 is responsible for the normal pattern generated in the presence of otherwise teratogenic amounts of RA.
How does Cyp26a1 protect the embryos from exogenous RA? We observe that, in embryos treated with 5 nM RA, cyp26a1 expression expands throughout the epiblast. This expanded expression presumably eliminates the excess RA and allows the normal onset of expression of cyp26b1, cyp26c1 and other redundant factors described above that can modulate RA-responsive gene expression in the hindbrain, and the hindbrain develops normally under these conditions. In cyp26a1 mutants treated with 5 nM RA, the excess RA is not eliminated, cyp26b1 and cyp26c1 expression is not initiated, and the entire brain is transformed to posterior hindbrain and/or anterior spinal cord identity. Unlike in untreated cyp26a1 mutants, cyp26b1 and cyp26c1 cannot compensate for the lack of cyp26a1 because they are not expressed.
The phenotype of cyp26a1-/- embryos treated with 5 nM
RA is significantly more severe than that of embryos depleted of all three
Cyp26 enzymes in the absence of exogenous RA. In the former, the entire brain
is transformed to an r7 and/or r8 identity whereas, in the latter, only the
hindbrain territory is transformed. This difference may be because, in
untreated embryos, RA simply does not diffuse as far as the midbrain, so Cyp26
enzymes are not required to inactivate it there. However, we have noted a
surprisingly sharp anterior limit of RA-responsiveness in Cyp26-depleted
embryos that corresponds with the posterior limit of the presumptive
cerebellum. It is possible that other mechanisms prevent RA signaling anterior
to this boundary. The development of the mid- and fore-brain has been shown to
require active repression of gene expression by unliganded RARs
(Koide et al., 2001), a
mechanism that is expected to be easily destabilized by the presence of RA. It
seems likely that multiple mechanisms exist that protect the mid- and
fore-brain from the teratogenic effects of RA.
Regulation of cyp26 expression
A major outstanding question is how is cyp26 expression normally regulated
in the hindbrain? Kudoh et al. (Kudoh et
al., 2002) showed that the normal posterior limit of
cyp26a1 in the hindbrain is established by signals (FGFs and Wnts)
from the margin because, in embryos treated with antagonists of these
pathways, the cyp26a1 boundary is shifted posteriorly. Although
cyp26a1 is directly inducible by RA, its anterior neurectodermal
expression arises independently of RA
(Dobbs-McAuliffe et al., 2004;
Sirbu et al., 2005) (our
work). The factors that positively regulate cyp26a1 in the anterior
neurectoderm remain to be identified.
We also do not know how cyp26b1 and cyp26c1 expression is
initiated in r2-r4, or what regulates their subsequent expansion into r5 and
r6. In general, the mechanisms controlling gene expression in the anterior
rhombomeres are poorly understood in any vertebrate
(Moens and Prince, 2002).
Similar to other anterior hindbrain genes, the initiation of cyp26b1
and cyp26c1 expression is independent of RA, because both genes are
expressed in DEAB-treated embryos. cyp26b1 and cyp26c1
expression is also independent of the prior establishment of hindbrain
boundaries by Cyp26a1, because both genes are expressed normally in
cyp26a1 mutants and in embryos in which cyp26a1 expression
is globally up-regulated by sub-teratogenic concentrations of RA. The early
expression domain of cyp26b1 and cyp26c1 in r3 and r4 is
similar to that of iro7, suggesting that they may be downstream of,
or co-regulated with, iro7
(Lecaudey et al., 2004).
Modern genetic and genomic resources available for the zebrafish will allow
the important mystery of cyp26 regulation to be addressed in the future.
Implications for the regulation of retinoic acid during hindbrain patterning
The model we propose for hindbrain patterning through localized RA
inactivation by Cyp26 enzymes (Fig.
7B) accounts for a number of previously unexplained aspects of
hindbrain patterning. First among these is the observation that embryos
depleted of endogenous RA can be rescued by exogenous RA. This rescue can be
achieved over a 20-fold range of RA concentrations, indicating that
RA-dependent gene expression is also not strictly concentration dependent. By
generating a stepwise pattern of RA degradation during hindbrain development,
Cyp26 enzymes eliminate the need for a continuous RA gradient. Secondly, a
major tenet of the RA morphogen model has been that more-posterior
RA-responsive genes, such as the hox-4 genes, are less sensitive to RA than
more-anterior ones, such as the hox-1 genes
(Gould et al., 1998); however,
this has recently been challenged by the observation that, in the context of
the intact enhancer, the RARE of hoxd4 is no less sensitive to RA
than the RARE of hoxa1 (Nolte et
al., 2003). Furthermore, posterior RA-responsive genes do not
require a longer exposure to RA than anterior ones, as has been proposed
(Dupe and Lumsden, 2001;
Sirbu et al., 2005), because
identical concentrations of RA applied shortly before the normal initiation of
expression are sufficient to rescue this expression in RA-depleted embryos,
irrespective of the anterior limit of the RA-responsive gene in question
(Maves and Kimmel, 2005).
According to our model, the anterior limit of hoxb1a, vhfn1 and
hoxd4 are determined not by different RA concentrations or length of
exposure of cells to RA, but simply by the posterior limit of Cyp26 activity
at the time of their expression onset. In its most extreme version, each
`step' in the model is essentially a binary decision in which cells posterior
to the Cyp26 domain experience RA and initiate RA-responsive gene expression
appropriate for that developmental time, while cells within the Cyp26 domain
do not. What determines which RA-responsive genes are available to be
expressed at a given time is the subject of ongoing studies. Recent work has
shown that, within a Hox cluster, the timing of hindbrain expression may be
regulated by the progressive opening of chromatin rather than the local
accumulation of active transacting factors
(Chambeyron et al., 2005).
A RA-dependent patterning mechanism that does not require the formation of a stable gradient in either space or time is expected to be robust to environmental fluctuations. RA is a potent teratogen that is derived from dietary sources of vitamin A, so a robust mechanism for controlling its activity is particularly important. This control does not appear to be exerted at the level of RA biosynthesis, because we have observed that, in the absence of Cyp26a1, even low amounts of precursor are highly teratogenic. Our step-wise model for hindbrain patterning by RA is robust in that it tolerates a broad range of environmental conditions.
Although our model provides robustness to the hindbrain-patterning process and explains how patterning can be established in the presence of uniform RA, our data does not rule out the possibility that other mechanisms act redundantly with RA degradation to pattern the hindbrain under normal circumstances. Indeed, our observation that hindbrain Cyp26b1 and Cyp26c1 activity is dispensable when global RA levels are kept in check by Cyp26a1 suggests that such mechanisms are at work. It is possible that a RA-responsive pre-pattern is established by a transient RAdiffusion gradient, but that Cyp26 enzymes are required to `lock in' this pattern. As with other developmental processes, it is likely that RA-dependent nervous system patterning events are controlled by overlapping, redundant mechanisms that modulate RA signaling at multiple levels. Our work demonstrates that Cyp26-dependent RA degradation is a crucial component of this complex regulation.
Note added in proof
Recently, Uehara et al. (Uehara et al.,
2006) reported an anterior expansion of retinoic acid
responsiveness in Cyp26a1-/-; Cyp26c1-/- double
mutant mice that is similar to the phenotype we have observed in
cyp26a1-/-; cyp26c1 MO-injected embryos, consistent with
an evolutionarily conserved role for Cyp26 enzymes in shaping retinoic acid
responsiveness in the hindbrain.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/1/02706/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Medical Scientist Training Program, University of
Wisconsin, Madison, WI, USA
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REFERENCES |
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J. Bowles and P. Koopman Retinoic acid, meiosis and germ cell fate in mammals Development, October 1, 2007; 134(19): 3401 - 3411. [Abstract] [Full Text] [PDF]
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L. L. Sandell, B. W. Sanderson, G. Moiseyev, T. Johnson, A. Mushegian, K. Young, J.-P. Rey, J.-x. Ma, K. Staehling-Hampton, and P. A. Trainor RDH10 is essential for synthesis of embryonic retinoic acid and is required for limb, craniofacial, and organ development Genes & Dev., May 1, 2007; 21(9): 1113 - 1124. [Abstract] [Full Text] [PDF]
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