1
MRC Intercellular Signalling Group, Centre for Developmental Genetics,
University of Sheffield School of Medicine and Biomedical Science, Firth
Court, Western Bank, Sheffield S10 2TN, UK
2
Department of Anatomy and Developmental Biology, University College London,
Gower Street, London WC1E 1BT, UK
3
Max-Planck Institut für Entwicklungsbiologie,
Spemannstrasse 36, 72076 Tübingen, Germany
*
Present address: Department of Biology, University of Konstanz, 78464
Konstanz, Germany
Present address: Department of Developmental and Cell Biology, University of
California at Irvine, Irvine, CA 92697-2300, USA
Author for correspondence (e-mail:
p.w.ingham{at}sheffield.ac.uk
)
Accepted 16 May 2001
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SUMMARY |
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Key words: Zebrafish, Anteroposterior patterning, Vitamin A deficiency, Retinoic acid, Retinoic acid receptor, Craniofacial development, Neural crest, raldh2, hoxb4
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INTRODUCTION |
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Retinoids are prime candidates for such posteriorising factors since they
can have a wide range of effects on AP-patterning in the developing central
nervous system (CNS), limbs and neural crest. Exposure of embryos to an excess
of retinoic acid (RA) inhibits anterior development in the neural tube and
craniofacial mesenchyme through the suppression of fore- and midbrain-specific
gene expression and the expansion of the expression domains of more caudally
restricted genes (reviewed by Durston et al.,
1998; Gavalas and Krumlauf,
2000
). These effects correlate
well with the distribution of endogenous RA: both in chick and mouse embryos,
RA is detected only after gastrulation with a sharp anterior boundary at the
level of the first somite, and at high concentrations caudal to this boundary
(Mendelsohn et al., 1991
;
Rossant et al., 1991
; Colbert
et al., 1995
; Horton and Maden,
1995
; Maden et al.,
1998
). Similarly, in zebrafish
the anterior trunk contains high levels of RA (Marsh-Armstrong et al.,
1995
).
Depriving embryos of RA causes a variety of developmental defects, among
them neural crest cell death, the absence of limb buds and posterior branchial
arches, small somites, and hindbrain segmentation defects, which collectively
are known as vitamin A-deficient (VAD) syndrome (Morriss-Kay and Sokolova,
1996; Maden et al.,
1996
; Dickman et al.,
1997
; Maden et al.,
2000
). In the hindbrain,
embryonic RA depletion leads to graded phenotypic effects: with decreasing
amounts of RA, expression of genes normally restricted anteriorly
progressively extends posteriorly until finally, in the absence of RA
signalling, embryos lack rhombomeric and gene expression boundaries posterior
to rhombomere 3 (Blumberg et al.,
1997
; Dickman et al.,
1997
; Dupe et al.,
1999
; Kolm et al.,
1997
; van der Wees et al.,
1998
; White et al.,
1998
; White et al.,
2000
).
The effects of RA and other retinoids are mediated through nuclear
receptors of the RAR and RXR families which act as ligand-activated
transcriptional regulators (reviewed by Mangelsdorf et al.,
1995). Inactivation of single
receptors in mice has revealed extensive receptor redundancy, while compound
mutations in some receptors recapitulate the phenotypic defects observed in
VAD, including the disruption of AP patterning in the cranial neural crest and
hindbrain (Dupe et al., 1999
;
Kastner et al., 1994
; Kastner
et al., 1997
). These complex
phenotypes are not surprising, given the widespread distribution of RA and
expression of its receptors. For example, zebrafish RAR
and
RAR
(rara and rarg Zebrafish
Information Network) expression show little overlap; RAR
is
expressed in paraxial mesoderm, posterior hindbrain and spinal cord, whereas
RAR
is expressed more anteriorly in head mesenchyme and in the
brain (Joore et al.,
1994
).
AP patterning of the CNS is mediated through the regulated expression of
Hox genes, which are expressed with discrete AP expression boundaries within
the developing neural tube and adjacent mesoderm. Binding sites for RA
receptors have been characterised in the regulatory regions of hoxa1,
hoxb1, hoxb4 and hoxd4, and shown to confer RA-mediated gene
activation in vivo and in vitro, suggesting that RA directly regulates Hox
gene transcription (Marshall et al.,
1994; Morrison et al.,
1996
; Dupe et al.,
1997
; Gould et al.,
1998
; Studer et al.,
1998
). Thus, the spatial
distribution of RA and its receptors are all thought to be critical for
regulating Hox gene expression in the neural tube.
The biosynthesis of RA involves the sequential conversion of vitamin A into
retinaldehyde, which is then oxidised to RA. At least two cytosolic alcohol
dehydrogenases (ADH), or microsomal retinol dehydrogenases, catalyse the first
step, while the second step requires cytosolic retinal dehydrogenases, members
of the aldehyde dehydrogenase (ALDH) family (reviewed by Duester,
2000). Two aldehyde
dehydrogenases, ALDH1 and ALDH6/V1, are predominantly expressed in spatially
restricted domains of the head and retina and are unlikely to contribute to
the high levels of RA posteriorly (Haselbeck et al.,
1999
; Maden et al.,
1998
). In contrast,
retinaldehyde dehydrogenase 2 (RALDH2), a nicotineamide adenine dinucleotide
(NAD)-dependent dehydrogenase, is expressed posteriorly in a pattern that
correlates with RA-mediated gene activation (Wang et al.,
1996
; Zhao et al.,
1996
; Niederreither et al.,
1997
; Berggren et al.,
1999
; Swindell et al.,
1999
). In mouse,
loss-of-function mutations in Raldh2 mimic the most severe phenotypes
associated with VAD, implicating Raldh2 as the main source of RA in
the vertebrate embryo (Niederreither et al.,
1999
; Niederreither et al.,
2000
).
We have characterised the neckless (nls) mutation in zebrafish, which recapitulates many aspects of VAD. We link nls to a missense mutation in raldh2, structural analysis of which predicts a non-functional protein. Consistent with the molecular nature of nls, we show that exogenous application of RA rescues the fin and mesodermal defects in nls mutants. We also show that zebrafish require raldh2 for formation of posterior head mesoderm and notochord, as well as for cell specification in the anterior spinal cord. Finally, we show that the lack of expression of hoxb4 in the CNS is due to defects in RA signalling from the paraxial mesoderm. Our findings suggest a model in which RA directs AP patterning directly in the mesoderm, and that these cells, in turn, indirectly pattern the neural tube.
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MATERIALS AND METHODS |
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Mutant screening
Diploid F2 progeny of male fish mutagenised with
ethyl-N-nitrosourea (ENU)(Mullins et al.,
1994; Solnica-Krezel et al.,
1994
) from a London wild-type
background (Currie et al.,
1999
) were fixed at 24 hours
postfertilisation (hpf) and hybridised with probes for krox20 (Oxtoby
and Jowett, 1993
),
pax2 (Krauss et al.,
1992
), shh (Krauss et
al., 1993
) and myoD
(Weinberg et al., 1996
). In
situ hybridisation was performed essentially as previously described (Begemann
and Ingham, 2000
), using
24-well plates. For double in situ hybridisations, strongly expressed
transcripts were labelled with fluorescein and detected with p-iodo
nitrotetrazolium violet (INT)/5-bromo-4-chloro-3-indolyphosphate (BCIP), and
weakly expressed ones were labelled with NBT/BCIP (Roche Diagnostics).
Mapping and linkage testing
nlsi26 was outcrossed to the WIK strain and the pooled
DNA from F2 homozygous mutants and sibling was analysed using
SSLPs. An EST (GenBank Accession Numbers, AI476832 and AI477235) that mapped
between z11119 and z8693 on the LN54 radiation hybrid panel (Hukriede et al.,
1999), was shown to encode
raldh2 by sequence similarity to other vertebrate Raldh2
genes. Linkage was determined by RFLP analysis of pooled cDNAs, from 40
`London wild type' and nls/nls embryos (oligonucleotides:
5'-AACTGCCAGGAGAGGTGAAGAACGAC-3' and
5'-ACGGCCATTGCCGGACATTTTGAATC-3'). PstI restriction of
the amplificates generated a restriction fragment length polymorphism (RFLP)
of 0.6 and 0.77 kb in nls/nls, and of 1.46 kb in wild type, owing due
to a missense mutation in nlsi26.
Cloning of raldh2
Degenerate primers against the peptide sequences IIPWNFP (5'-ATA/C/T
ATA/C/T CCI TGG AAC/T TTC/T CC-3') and PFGGFKM (5'-CAT C/TTT A/GAA
ICC ICC A/GAA IGG-3') were used to amplify a 0.9 kb raldh2
fragment by RT-PCR from 30 hours hpf wild-type embryos. Fragments were
subcloned into the pCR2.1-vector using the TOPO kit (Invitrogen) and
sequenced, revealing one with similarity to vertebrate Raldh2. The
fragment was screened against a zebrafish late somitogenesis stage cDNA
library (Max-Planck-Institute for Molecular Genetics, Berlin) under stringent
conditions to obtain a full-length clone of raldh2
(ICRFp524L2053Q8)(GenBank Accession Number, AF339837). Several cDNAs from
different homozygous nls mutant embryos and London wild-type embryos
were obtained by RT-PCR and sequenced using raldh2-specific
primers.
Retinoic acid treatments
Batches of 60-80 embryos from wild-type or nls heterozygous
parents were incubated in the dark from late blastula stage onwards in varying
dilutions (in embryo medium) of a10-4 M all-trans RA
(Sigma)/10% ethanol solution (from a 10-2 M stock solution in
DMSO). As controls, siblings were treated with equivalent concentrations of
ethanol/DMSO alone. Mild teratogenic effects (e.g. disrupted heart development
and smaller eyes) were observed at higher concentrations.
mRNA rescue experiments
Full-length RALDH2 cDNA was cloned as a SpeI/NotI
fragment into the XhoI and NotI sites of pSP64TXB (Tada and
Smith, 2000). The resulting
plasmid, pSP64T-RALDH2 was linearised with XbaI and transcribed using
the `SP6 mMessage mMachine' kit (Ambion). 3 nl of in vitro synthesised mRNA
was injected into embryos at the one-to four-cell stage.
Morpholino injections
Two partially overlapping morpholinos against raldh2 (5'-gtt
caa ctt cac tgg agg tca tc-3' and 5'-gca gtt caa ctt cac tgg agg
tca t-3') were obtained from GeneTools, LLC, and solubilised in water at
a stock concentration of 1 mM (8.5 mg/ml). 4-5 nl of 1:2, 1:4 and 1:10
dilutions in water, respectively (approximately 4, 2 and 0.85 mg/ml) were
injected into one-cell stage embryos. The injected dilutions resulted in
strong (1:2) to weak (1:10) phenocopies of the nls phenotype.
Histology
Cartilage staining was performed as described (Schilling et al.,
1996). TUNEL staining for
apoptotic cells was performed as described previously (Williams et al.,
2000
). Labelled DNA was
detected with alkaline phosphatase-coupled anti-fluorescein (Roche), followed
by a NBT/BCIP (Roche) colour reaction. For live labelling of apoptosis,
dechorionated embryos were incubated in 5mg/ml Acridine Orange (Sigma)/1%
DMSO/PBS, washed in PBS and observed with a fluorescein filter set.
Immunostaining was carried out according to Westerfield (Westerfield,
1995
) with antibodies against
No tail (Schulte-Merker et al.,
1994
), myosin heavy chain
(Dan-Goor et al., 1990
) and
the cell-surface protein DM-GRASP (Zn8; Trevarrow et al.,
1990
). Embryos were cleared in
70% glycerol, mounted on bridged coverslips and photographed on a Zeiss
Axioplan microscope.
Mosaic analysis
Donor embryos were injected at the one-cell stage with 2.5% lysine fixable
tetramethyl-rhodamin-dextran and 3.0% lysine fixable biotindextran
(Mr 100.000)(Molecular Probes) dissolved in 0.2 M KCl.
Transplants were done blindly, and donor genotypes determined at 24 hpf. At
late blastula stages, groups of 10-30 donor cells were transplanted into
unlabelled host embryos of the same stage and placed either along the margins
of the blastoderm, which gives rise to the mesendoderm, or further away from
the margin in regions that give rise to neural ectoderm (Kimmel et al.,
1990). Transplanted cells were
labelled using a peroxidase-coupled avidin (Vector Labs) and detected with
diaminobenzidine (for brightfield microscopy) or a fluorescent tyramide
substrate (Renaissance TSA kit; Dupont Biotechnology Systems), and examined
for fluorescence.
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RESULTS |
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To investigate its mesodermal defects further, we compared markers of
different mediolateral regions (paraxial, lateral plate and axial). Analysis
of myoD and her1 expression revealed no differences in the
length of the somitic plate, or number of somites formed, in nls
homozygotes (Fig. 1G-J). The
nephric tubules, which derive from lateral plate mesoderm and express
pax2, are invariantly located lateral to somite 3 in nls, as
in wild type (Fig. 1K,M). By
contrast, nls mutants have fewer notochord cells, visualised using an
anti-No tail antibody in the posterior head
(Fig. 1G-J; see
Table 1). At 12 hpf the number
of No tail-positive cells between r5 and somite 1 is reduced by approximately
50% in nls (Table 1).
The number of developing anterior somites is unchanged, as expression of
hoxc6 is detected up to the boundary between the fourth and fifth
somites in nls as in wild type (Molven et al.,
1990; Prince et al.,
1998a
). Thus, nls
mutants exhibit early defects in both paraxial and axial mesoderm in the
region that will form the posterior head and pectoral fins.
|
A mutation in raldh2 co-segregates with nls
Using bulked segregant analysis we mapped nls between SSLPs z11119
and z8693 on LG7 (Fig. 2B,C).
This location coincides with that of an EST predicted to encode a close
relative of mammalian Raldh2. As the nls phenotype shows
some similarities to that of VAD quail and Raldh2 mutant mouse
embryos, Raldh2 seemed a good candidate for the nls gene. We
sequenced a full-length cDNA encoding zebrafish raldh2
(Fig. 2A) and six independent
isolates of RALDH2 cDNAs from homozygous nls embryos that all contain
a point mutation (Gly204Arg) (Fig.
2D). This creates a fortuitous PstI restriction site with
which we confirmed linkage to nls by RFLP analysis
(Fig. 2E). Glycine 204 is one
of 23 residues that are invariant among 16 NAD and/or NADP-linked aldehyde
dehydrogenases with wide substrate preferences, as well as types with distinct
specificities for metabolic aldehyde intermediates, particularly semialdehydes
(Perozich et al., 1999) and
forms the core of a loop-forming sequence motif that lies within the
NAD-binding domain of the molecule. Modelling the structural effects of
substituting glycine 204 with arginine by comparison with the tertiary
structure of rat RALDH2 (Lamb and Newcomer,
1999
) suggest that this
mutation prevents a secondary structure that allows interaction of the protein
with the co-enzyme NAD (data not shown). Owing to the tight spacing of glycine
204 within its surroundings, replacing this residue with arginine appears to
be sterically prohibitive and would create a protein of reduced or no
activity.
|
Morpholino-mediated translational inhibition of RALDH2 phenocopies
nls
To investigate whether loss of RALDH2 activity could account for the
nls phenotype, we injected raldh2-specific morpholino
antisense oligonucleotides into wild-type embryos and assayed the ensuing
effects by in situ hybridisation with appropriate marker probes. Injection of
either 8.5 or 17 ng of the raldh2-morpholino resulted in a strong
reduction in the space between the krox20 and myoD
expression domains at 12 hpf relative to wild type
(Fig. 3A), a phenotype
indistinguishable from that of nlsi26 embryos at the same
stage (Fig. 1I). On average, 71
out of 101 embryos injected with both concentrations and either morpholino
exhibited this phenotype. Moreover, distinct rhombomeres r3 and r5 can be
observed in the injected embryos. At 24 hpf, the post-otic head is shortened
and tbx5 expression in the pectoral fin buds is abolished (not
shown). We never observed phenotypes stronger than those exhibited by
nlsi26 homozygotes, indicating that the
nlsi26 mutation is equivalent to the loss of RALDH2
activity. Injection of 34 ng raldh2-morpholino did, however, cause
neural necrosis, which we interpret to be a nonspecific effect.
|
Exogenous RA or RALDH2 activity rescues aspects of the nls
mutant phenotype
As RALDH2 catalyses the last step in the synthesis of
all-trans-RA, the main constituent of retinoids in zebrafish embryos
(Costaridis et al., 1996), we
investigated whether or not exogenous RA can rescue the mesodermal and fin
defects caused by nls. Two early fin markers, tbx5.1, which
labels the entire pectoral fin field (Begemann and Ingham,
2000
), as well as shh,
a marker of posterior fin mesenchyme (Krauss et al,
1993
), are undetectable in the
presumptive fin mesenchyme of nls homozygotes
(Fig. 3C,E,G-I). Exposure to
all-trans-RA rescued caudal head mesoderm development at 12 and 17
hpf (Fig. 3B;
Table 2), as well as the
pectoral fin expression of tbx5.1 at 36 hpf
(Fig.3J,L), consistent with the
nls mutation causing a reduction or loss of RA signalling.
|
To confirm that the molecular lesion in nls/raldh2 is responsible for the nls mutant phenotype, we injected in vitro transcribed raldh2 mRNA into one- to four-cell nls embryos and assayed for the rescue of tbx5.1 expression in the pectoral fin buds, as well as for development of an apical fin fold. The concentration of injected raldh2 mRNA was progressively reduced until overexpression phenotypes, similar to those observed in RA-treated embryos, were no longer observed. This concentration (approx. 500 pg per embryo) was used to assay phenotypic rescue in batches of embryos derived from a cross between nls-heterozygotes (Table 3). Partial or complete restoration of tbx5.1 expression was seen in 83% of mutants (30/36 expected nls embryos), indicating that wild-type raldh2 is sufficient to rescue nls embryos.
|
nls/raldh2 is expressed in early trunk paraxial
mesoderm
To investigate the spatial and temporal patterns of nls/raldh2
expression during embryogenesis, we used whole-mount in situ hybridisation.
raldh2 mRNA is first detectable at 30% epiboly in an open ring along
the blastoderm margin (Fig.
4A). Upon gastrulation, raldh2 is expressed in involuting
cells at the margin that will form mesendoderm
(Fig. 4B,C), but is excluded
from the most dorsal cells of the embryonic shield. Expression persists in
posterior and lateral mesoderm during gastrulation and remains excluded from
notochord precursors (Fig. 4D).
By 15 hpf, expression is found in forming somites, as well as in lateral plate
mesoderm extending into the cranial region and in the pronephric anlage
(Fig. 4E). Somite expression
persists throughout segmentation (Fig.
4F,G,I,J) becoming progressively restricted to the somite
periphery. By 32 hpf, nls/raldh2 is expressed in subsets of the
pharyngeal arch mesenchyme adjacent to the otic vesicle
(Fig. 4H,K) and in the
posterior mesenchyme of the forming pectoral fins
(Fig. 4N). Other sites of
expression are the endoderm (not shown), cells in somites 1-3 adjacent to the
notochord and spinal cord (Fig.
4L), the dorsal retina and choroid fissure
(Fig. 4O), and motoneurones
that innervate the pectoral fins (Fig.
4M and data not shown). Surprisingly, we found that in
nls embryos the expression of nls/raldh2 is upregulated in
somites and in the cervical mesoderm that flanks the posterior hindbrain
(Fig. 4P-S), whereas expression
is absent in structures that are reduced in the mutant (see below).
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Craniofacial skeletal and muscle defects in nls
To determine the later consequences of the embryonic patterning defects in
nls for larval development, we examined skeletal and muscle anatomy.
In all vertebrates, cells of the cranial mesoderm give rise to the pharyngeal
and limb musculature (Noden,
1983; Schilling and Kimmel,
1994
), which express the
myogenic marker myosin heavy chain (Fig.
5A-D). In zebrafish larvae at 72 hpf, pharyngeal muscles can be
identified by their segmental attachments and positions along the dorsoventral
axis within each pharyngeal arch (Schilling and Kimmel,
1997
). nls mutants
develop normal patterns of muscles in the first two arches, the mandibular and
hyoid, as well as extraocular muscles, while muscles of the five branchial
arches (i.e. dorsal pharyngeal wall muscles, rectus ventralis, transversus
ventralis), which derive from posterior head mesoderm, are absent
(Fig. 5B,D). Consistent with
the molecular data indicating that the identities of anterior somites are
unaffected in nls, we found that the sternohyal muscles, which are
thought to originate from myoblast populations within the somites 1-3
(Schilling and Kimmel, 1997
),
are present (Fig. 5D).
|
Defects in the branchial musculature in nls mutants correlate with defects in the neural crest-derived head skeleton, which can also be identified by their segmental locations (Fig. 5E,F). Alcian Blue staining showed that cartilages of the mandibular and hyoid arches are present in nls, though reduced in size compared with wild type. In contrast, skeletal elements in the branchial arches are reduced or absent (from dorsal to ventral these include the ceratobranchials and hyobranchials in arches 4-7, and an axial row of basibranchials in arches 4 and 5) whereas these elements are still present in branchial arch 1. The expressivity of this phenotype is dependent on the genetic background, so that in individual outcrossed lines of nlsi26 all five branchial arches may be deleted. All cartilage elements of the pectoral skeleton are also consistently absent. The defects in branchial arch morphogenesis in nls are mirrored by the lack of formation of endodermally derived pharyngeal pouches that form the prospective gill slits (Fig. 4G,H). Thus, the early defects at the head/trunk boundary during somitogenesis in nls correlate with later defects in the formation of tissues derived from all three germ layers in the pharyngeal segments as well as the pectoral fins that form in this location.
Neural crest defects in nls mutants
To investigate the embryonic basis of defects in the neural crest-derived
cartilages of the larva, we analysed markers of both premigratory and
migrating neural crest populations. We found no differences in the expression
of markers of premigratory crest in nls, such as snail2,
which marks most neural crest cells at 12 hpf (data not shown) or
krox20, which marks a small group of cells that emigrate from r5 at
13 hpf (see Fig. 1H,J). In
contrast, expression of dlx2, which marks all three migrating streams
of neural crest and persists in the arch primordia, is disrupted specifically
in the most posterior stream that will form the branchial cartilages.
Precursors of the mandibular and hyoid arches appeared to migrate normally
(Fig. 5I,I') and
dlx2 expression in these arches appeared only slightly reduced by 40
hpf (Fig. 5J,J'). To test
the possibility that the branchial neural crest cells undergo apoptotic cell
death, we labelled dying cells in nls mutants with whole-mount TUNEL
staining or Acridine Orange (Fig.
6C,D). At 24 hpf, we observed increased cell death in nls
mutants in the anterior notochord and the third and fourth branchial arches
(Fig. 5K',L'),
indicating that survival of posterior branchial neural crest cells requires
nls.
Hindbrain defects in nls mutants
To investigate whether the mesodermal defects in nls are
accompanied by defects in the neurectoderm, we examined the expression of a
number of genes that mark specific AP regions of the hindbrain. Expression of
krox20, which marks r3 and r5, is initially weaker in r5 at tailbud
stage, but subsequently becomes indistinguishable from wild type
(Fig. 1G-J). Expression of
valentino (Moens et al.,
1998), which marks r5/r6, is
expanded by 30 µm along the AP axis in nls mutants
(Fig. 6A,B). Likewise,
eph-b2 expression which marks r7 (Durbin et al.,
1998
) is slightly expanded in
nls as compared to wild type between 14-15 hpf
(Fig. 6C,D). Thus, posterior
rhombomere territories appear to be established in the appropriate locations
in nls mutants, but are slightly enlarged relative to their wild-type
counterparts. Consistent with this, we found that hoxb3, which in
wild type is strongly expressed in a stripe that includes r5/r6, is expressed
in a similar but expanded r5/r6 domain in nls mutants
(Fig. 6E-H).
In contrast, we found a much stronger defect in hoxb4 expression,
which in wild-type extends throughout the posterior neurectoderm up to a
boundary between r6 and r7 (Prince et al.,
1998a). In nls
embryos, hoxb4 expression cannot be detected in this region before
15-16 hpf, although it is expressed normally in the somitic mesoderm and in
the tailbud (Fig. 6I,J; and not
shown). By 16 hpf, however, hoxb4 expression is established at a more
or less appropriate position in the neural tube, although it does not extend
caudally towards the tailbud (Fig.
6K,L).
RARs are autonomously required for the neural induction of hoxd1
by mesodermal signals in in vitro conjugates from Xenopus, while in
the chick, Hoxb4 is a direct target of RAR (Kolm et al.,
1997
; Gould et al.,
1998
). To test if the defect
in hoxb4 expression in nls might be due to a disruption in
RAR expression, we analysed the distribution of both RAR
and
RAR
mRNA (Joore et al.,
1994
). We detected no defects
in RAR
in nls (not shown); however,
RAR
expression is downregulated precisely in the region of the
neural tube disrupted in mutants. By contrast, RAR
expression
outside the CNS in the mesoderm appears to be slightly upregulated, and
expression in the tailbud is unaffected
(Fig. 6M,N). Thus, defects in
RAR
regulation in nls correlate with the defects in
expression of hoxb4.
To examine the consequences of these changes in gene expression for
neuronal patterning in the posterior hindbrain we used a combination of
neuronal markers and dye labelling techniques. A subset of interneurones
express tbx-c (Dheen et al.,
1999). In nls,
expression in these interneurones is strongly reduced at 36 hpf
(Fig. 6P,R). We also
retrogradely labelled the large primary reticulospinal interneurones of the
hindbrain with rhodamine-dextran by injection into the spinal cord; these are
variably disrupted in the caudal hindbrain of nls mutants (data not
shown). Likewise, spinal motoneurones that innervate the pectoral fin bud are
reduced in nls, as revealed by labelling with the Zn8 antibody
(Fig. 6S,T). The loss of
hindbrain interneurones, as well as neuronal subpopulations in the rostral
spinal cord, correlates with the restricted defects in gene expression at this
AP level, such as the failure to initiate hoxb4 expression. Thus,
neural defects are milder compared with complete loss of RA signalling, as in
the Raldh2-/- mouse, where the caudal hindbrain is absent
due to misspecification to a more rostral fate.
RALDH2 is required in the mesoderm for initiation of hoxb4
expression in neural ectoderm
To determine the cellular requirement for nls function, we
transplanted cells from embryos labelled with a lineage tracer into unlabeled
hosts at the gastrula stage (Fig.
7). First, we asked if nls cells can contribute to
tissues disrupted in the nls mutation, such as the posterior head
mesoderm, and axial, paraxial and posterior hindbrain. In an otherwise
wild-type embryo, donor derived nls cells were found to be able to
spread widely throughout the neural ectoderm of the hindbrain and anterior
spinal cord (Fig. 7A,B,I; Table 4). In other cases,
mutant cells readily populated regions of the paraxial
(Fig. 7A') and axial
(Fig. 7C) mesoderm of the head
(Table 4). Likewise,
transplants of wild type mesoderm into nls mutants were able to
populate the anterior somites (Fig.
7F).
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We then used such mesodermal grafts to determine whether the defects in
hoxb4 expression in the neural tube of nls mutants
(Fig. 7D,E) might reflect
defects in a non-autonomous signal from surrounding mesoderm that requires
nls/raldh2. With reference to fate maps of head mesoderm (Kimmel et
al., 1990), we transplanted
mesodermal precursors from biotin-dextran labelled wild-type donors into
unlabeled mutant hosts at the gastrula stage. In many cases these transplanted
cells spread widely along one side of mutant host embryos and often form
muscles in the anterior somites adjacent to the region in which hoxb4
is normally expressed (Fig.
7F-H). In 71% of cases in which wild-type cells populated this
region in nls, we observed a partial recovery of early hoxb4
expression several hours before 15 hpf
(Table 4). Control transplants
of mutant mesoderm into nls hosts had no such effect on
hoxb4 expression (Fig.
7G). Thus, the activity of nls/raldh2 in paraxial
mesoderm is necessary for hoxb4 expression in the adjacent neural
ectoderm.
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DISCUSSION |
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The combination of hindbrain, neural crest and limb defects characteristic
of nls mutant embryos is similar to that caused by targeted
inactivation of Raldh2 in the mouse (Niederreither et al.,
1999), as well as by VAD in
the quail (Maden et al., 1996
;
Gale et al., 1999
) and rat
(White et al., 2000
) embryos.
The hindbrain defects in nls embryos are, however, less severe than
in these other cases: in both the mutant and VAD embryos, rhombomere-specific
characteristics caudal to r4 are disrupted whereas in nls, posterior
rhombomeres appear slightly expanded and only neurones near the
hindbrain-spinal cord boundary are disrupted. This phenotype is reminiscent of
the milder forms of VAD in rat embryos (White et al.,
2000
) and of the partial
rescue of Raldh2-/- mice by maternal application of RA
(Niederreither et al., 2000
),
and suggests that the posterior hindbrain and anterior spinal cord are most
sensitive to a reduction in RA levels.
The fact that the nls phenotype is closer to the effects of
attenuation, rather than elimination of RA signalling in amniote embryos could
be explained if the nlsi26 allele behaves as a hypomorph,
the mutant protein retaining residual enzymatic activity. Against this,
however, structural modelling predicts that the glycine-to-arginine
substitution found in nlsi26 would result in a complete
loss of activity, a view supported by our finding that nls is
precisely phenocopied by morpholino-mediated translational inhibition of the
raldh2 gene that we have cloned. This raises the possibility that
zebrafish may possess a second raldh2 gene that can partially
compensate for the loss of nls/raldh2, a possibility consistent with
the finding that many teleost genes are duplicated (Amores et al.,
1998).
A restricted requirement for nls/raldh2 at the head/trunk
boundary
RA has been proposed to act as a graded posteriorising signal throughout
the AP axis of the CNS (reviewed by Gavalas and Krumlauf,
2000). Mutation of
nls/raldh2 and reduction of RA in zebrafish through pharmacological
inhibition of aldehyde dehydrogenases (Perz-Edwards et al.,
2001
), however, suggest that
RA acts in a more localised manner. From tail bud stages onwards,
nls/raldh2 expression is confined to trunk and tail mesoderm, yet
defects in nls are largely in the posterior head. Thus,
nls/raldh2 and, by inference, RA produced in presumptive somites may
act at only a short distance and at high concentrations. Perhaps only cells in
close proximity to the source of RA are able to respond, while others require
different posteriorising signals, such as members of the fibroblast growth
factor and Wnt families.
Defects in paraxial mesoderm in nls/raldh2 mutants may secondarily
cause its hindbrain defects through loss of local posteriorising induction
(Itasaki et al., 1996).
nls mutants lack mesoderm between the level of r5 and somite 1 at the
beginning of somitogenesis, suggesting that nls/raldh2 activity must
be required during gastrulation; this correlates well with the early zygotic
expression of nls/raldh2 in the germ ring of gastrulating embryos,
which forms the mesendoderm (Kimmel et al.,
1990
). In VAD quail embryos, a
similar defect has been accounted for by apoptosis of mesodermal cells within
the first somite during a brief period in early somitogenesis (Maden et al.,
1997
). Such patterns of
apoptosis were not observed, however, during gastrulation stages in
nls. Moreover, the mesodermal deficiency in nls/raldh2
occurs in a much broader region anterior to the somites, and includes both
axial and paraxial cells. Our molecular analysis rules out the possibility
that the posterior head mesoderm is transformed into more caudal tissues, as
anterior-most somites in nls develop with their normal identities. RA
may instead be involved in maintaining cell proliferation at the head/trunk
boundary, though we currently have no direct evidence for such an effect.
Surprisingly, we find defects not only in the paraxial mesoderm of
nls, but also in the notochord, a structure that is known to
influence patterning of the overlying neural tube. Although the notochord has
not been implicated specifically in AP patterning of the hindbrain, recent
evidence in zebrafish has shown that some Hox genes are expressed in AP
restricted domains near the head/trunk boundary (Prince et al.,
1998b). nls/raldh2 is
not expressed in the notochord, suggesting that the effects on its development
are non-autonomous, which is supported by our mosaic results. Our data suggest
that, similar to its restricted influences on the posterior hindbrain, RA
signalling from the somites acts locally on axial as well as paraxial
mesoderm.
Correlated with these spatially restricted phenotypes in the mesoderm and
nervous system, nls mutants later exhibit defects in branchial
arches. Again these are confined to the posterior head and recapitulate the
branchial hypoplasia in chick embryos treated with pan-RAR antagonists and in
Raldh2-deficient mice (Niederreither et al.,
1999; Wendling et al.,
2000
). In this case, however,
Raldh2 appears to be required for cell survival in the neural
crest-derived skeleton. Crest cells are apoptotic in the pharyngeal primordia
in nls, as they are in VAD and Raldh2-/- mice
(Maden et al., 1996
;
Niederreither et al., 1999
).
The correlation between the loss of posterior head mesoderm and posterior
arches in nls mutants further suggests that apoptosis may be a
secondary consequence of earlier defects in posterior head mesoderm and/or
endoderm. Patterning of cranial neural crest has been shown to respond to both
mesenchymal-mesenchymal interactions with mesoderm, as well as
epithelial-mesenchymal interactions with surrounding endoderm in the arches
(Trainor and Krumlauf, 2000
;
Tyler and Hall, 1977
).
Alternatively, crest cells may require nls/raldh2 directly for their
survival, as crest cells appear to be particularly sensitive to alterations of
RA levels (Ellies et al.,
1997
). RAR
/RARß
double mutant mice have hypoplastic posterior branchial arches similar to
those seen in ablations of postotic neural crest in chick embryos (Dupe et
al., 1999
; Ghyselinck et al.,
1997
), despite the normal
generation and migration of crest. Thus, the developmental deficiencies
observed in the pharyngeal region of nls are likely to be caused by
local defects in postotic mesoderm and endoderm, rather than by a long-range
graded requirement for RA.
Another striking aspect of the nls phenotype is the complete lack
of pectoral fin buds. nls/raldh2 is required early during pectoral
fin induction locally in the fin field, as one of the earliest markers of the
fin field, tbx5.1, is not expressed in the lateral plate mesoderm in
nls mutants. This phenotype correlates well with expression of
nls/raldh2 in this region of the mesoderm between the 6- and
12-somite stage (12-15 hpf). raldh2 expression precedes (not shown)
and is then maintained during outgrowth of the apical fold at the posterior of
the pectoral fin bud (Fig. 3R).
As we have shown for hoxb4 in the neural tube, nls/raldh2
may also be required for the expression of Hox genes in the prospective fin
field, thus being involved in setting up limb position along the AP axis of
the lateral plate mesoderm (Cohn et al.,
1997). A common model of
limb-field determination proposes a function for fibroblast growth factors
(FGFs) as limb inducers and locates the source of limb inducing activity in
the intermediate mesoderm (reviewed in Martin,
1998
). RA induces FGF or
generates competence of the flank to respond, and FGFs are capable of inducing
ectopic limb buds in the lateral plate mesoderm of the chick flank.
raldh2 may thus be required for the local induction of an FGF or
another inducing signal.
The role of the mesoderm in neural patterning
A large body of evidence has previously implicated RA in mediating signals
from the mesoderm to the neural tube. In Xenopus, tissue
recombination experiments have demonstrated a requirement for RARs in the
ectoderm for induction of Hox gene expression through mesoderm-derived signals
(Kolm et al., 1997).
Conversely, upregulation of RA during somitogenesis has been shown to be
required for cultured paraxial mesoderm to induce cells of spinal cord fate,
while loss of this inducing capacity by freeze-thawing paraxial cells can be
restored through administration of RA (Muhr et al.,
1999
). In line with these
data, transgenic RARß-lacZ reporter constructs (where the
RA-responsive element of the RARß-gene has been fused to the
lacZ reporter gene) (Balkan et al.,
1992
; Mendelsohn et al.,
1991
; Rossant et al.,
1991
; Zimmer,
1992
), as well as similar
reporter constructs in zebrafish (Marsh-Armstrong et al.,
1995
; Perz-Edwards et al.,
2001
), are activated in the
neural tube. Such activation of neural RA-responsive genes can be explained by
diffusion of RA from the paraxial mesoderm. While the lack of detectable
nls/raldh2 mRNA in the prospective neural tube during gastrulation
and segmentation stages is highly suggestive of a non-autonomous action of RA,
the results of our mosaic analyses provide the first conclusive evidence for
this mode of action. Transplantation of wild-type cells into the somitic
mesoderm of nls embryos restores early hoxb4 expression,
whereas that of nls cells in the same position does not. Likewise,
nls ectodermal cells intercalate normally into a wild-type CNS in the
hoxb4-expressing domain, suggesting that nls/raldh2 is not
required for cells to take on the identity of this region of the neural
tube.
Our analysis of hoxb4 expression in nls reveals that, as
in the amniote embryo (Gould et al.,
1998), the zebrafish
hoxb4 gene follows a biphasic mode of transcriptional regulation. The
first phase establishes neural expression and depends on raldh2
activity, while the second phase is independent of raldh2. These
regulatory steps are linked to neural promoter elements that regulate
hoxb4 expression in chick and mouse embryos, one of which acts before
rhombomere formation and one of which acts later in maintenance of expression.
Activation of the early neural enhancer is mediated by RA response elements
(Gould et al., 1998
). Our
results are consistent with a similar control of hoxb4 expression in
fish: hoxb4 is initially not expressed but recovers in
nls/raldh2 mutants after 15-16 hpf, albeit to less than its full
posterior extent. The loss of hindbrain interneurones and neurones in the
anterior spinal cord may result from a failure to initiate this first phase of
hoxb4 expression. Similarities in the expression of
RAR
in the neural tube (Joore et al.,
1994
) and dependence on
raldh2 suggests that RAR
is likely to be a major
effector of paraxial RA signalling in the neural tube. This is supported by
the finding that overexpression of a dominant negative form of
Xenopus RAR
1 in the avian neural tube blocks induction of
hoxb4 (Gould et al.,
1998
). RAR
expression may be under autoregulatory control during this phase or may be
controlled by other RARs, as its neural expression is dependent upon
RA signalling. Similarly, nls/raldh2 itself may be subject to a
RA-mediated autoregulation within the somitic mesoderm, as its mRNA levels
increase in nls/raldh2 mutant embryos older than 30 hpf. In line with
this, previous studies in chick have shown that exogenous RA represses
raldh2 expression.
Our model of short-range RA-dependent interactions between the mesoderm and neural tube (Fig. 8) based on our genetic analysis in zebrafish is consistent with earlier results obtained from experimental manipulation of the chick embryos. This model makes testable predictions and thus provides a framework for future experiments, both to explore the exact source and graded nature of the RA signal and the nature of the responses of neural cells to RA during vertebrate development.
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ACKNOWLEDGMENTS |
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