1 Howard Hughes Medical Institute and Division of Basic Sciences, Fred
Hutchinson Cancer Research Center, 1100 Fairview Avenue North, PO Box 19024,
Seattle, WA 98109, USA
2 Medical Scientist Training Program and Molecular and Cellular Biology Program,
University of Washington, Seattle, WA 98195, USA
Author for correspondence (e-mail:
cmoens{at}fhcrc.org)
Accepted 8 June 2004
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SUMMARY |
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Key words: Rhombomere, Hindbrain, Retinoic acid, vhnf1, valentino, mafB, Fibroblast growth factor, hox
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Introduction |
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Previous work has shown that the transcription factors MafB and Krox20 are
direct regulators of Hox gene expression in r5 and r6. Kreisler
(Kr) and valentino (val), the mouse and zebrafish
orthologs of mafB, respectively, are required for normal r5 and r6
development (Cordes and Barsh,
1994; McKay et al.,
1994
; Moens et al.,
1996
; Moens et al.,
1998
; Prince et al.,
1998
) and MafB has been shown to activate hoxb3 and
hoxa3 expression directly in transgenic mice
(Manzanares et al., 1997
;
Manzanares et al., 1999a
;
Manzanares et al., 2001
;
Manzanares et al., 2002
).
Krox20 is required for the maintenance of r3 and r5 identity
(Schneider-Maunoury et al.,
1997
) and cooperates with Kr to regulate hoxb3 directly
in r5 (Manzanares et al.,
2002
). Furthermore, hox3 genes are both necessary and
sufficient for the development of somatic motoneurons in the hindbrain
(Gaufo et al., 2003
;
Guidato et al., 2003
).
Since mafB/Kr/val plays an important and conserved role in the
control of r5 and r6 specification, significant effort has been made to
understand how its expression is established in the developing hindbrain.
Several inputs onto mafB/Kr/val expression have been identified:
retinoic acid (RA) signaling is necessary for acquisition of all hindbrain
fates posterior to r3, including expression of mafB/Kr/val, as
determined by pharmacologic and genetic disruption of RA production or
activity (Dupe and Lumsden,
2001; Gavalas and Krumlauf,
2000
; Linville et al.,
2004
; Wendling et al.,
2001
). Transplantation and genetic mosaic analyses have suggested
that the relevant source of RA for posterior hindbrain patterning appears to
be the trunk paraxial mesoderm (Begemann et
al., 2001
; Gould et al.,
1998
). Secondly, variant hepatocyte nuclear factor1
(vhnf1; tcf2 Zebrafish Information Network), a
homeodomain transcription factor expressed throughout the posterior hindbrain
and anterior spinal cord, was identified in a genetic screen in the zebrafish
as a positive regulator of val
(Sun and Hopkins, 2001
).
Finally, work in zebrafish showed that r4 is a source of Fgf3 and Fgf8, which
are required for the patterning of surrounding rhombomeres, including the
initiation of val expression
(Maves et al., 2002
;
Walshe et al., 2002
).
Wiellette and Sive (2003
) have
recently demonstrated that Fgfs and Vhnf1 synergize to drive the expression of
both val and krox20 in r5.
Specification of r5 and r6 identities requires not only the activation of
r5- and r6-specific gene expression, but also the repression of r4-specific
gene expression. Recent work in the zebrafish has shown that Vhnf1 represses
hoxb1a in a val-independent manner
(Wiellette and Sive, 2003),
while other work in the mouse has shown that hox3 genes, which are
targets of MafB/Kr/Val, are required for hoxb1 repression
(Gaufo et al., 2003
). The role
of MafB/Kr/Val itself in the repression of hoxb1 is controversial:
while some posterior expansion of hoxb1 expression in mouse
Kr mutants and zebrafish val mutants has been reported
(McKay et al., 1994
;
Prince et al., 1998
), little
expansion of a Hoxb1-r4 reporter transgene was observed in
Kr mutants (Manzanares et al.,
1999b
).
We have investigated how global RA signals, local FGF signals and vhnf1 expression are integrated to specify r5 and r6 in the developing zebrafish hindbrain. We show that RA signals are essential for the activation of vhnf1 expression and that Vhnf1 acts downstream of RA signaling to drive val expression. Secondly, we show that Vhnf1 strictly requires r4-derived Fgf signals, probably through the Map kinase cascade, to initiate val expression. Vhnf1 therefore integrates local r4-Fgf signals with global positional information provided by RA to specify r5 and r6 identities. We have also investigated how Vhnf1 and Val contribute to the repression of r4-specific gene expression in the r5-6 territory. We find that repression of r4 gene expression in the r5-6 territory is initially established in a vhnf1-independent manner, but that vhnf1 is then rapidly required to reinforce this restriction. By contrast, val plays a relatively minor and late role in the repression of hox1/r4-identity. Thus, specification of r5 and r6 identity is achieved through temporally and genetically distinct steps that establish and then maintain repression of r4 identity, as well as activating r5- and r6-specific gene expression. Furthermore, we find that different aspects of rhombomere identity, specifically those determining neuronal identity and those determining cell-surface character, are regulated independently. As a consequence, cells with very different `hox codes' can mix freely in genetic mosaics because they share an `Eph-ephrin code.'
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Materials and methods |
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Two color in RNA in-situ hybridization was performed essentially as
described (Prince et al.,
1998), except that in some cases BM-Purple (Roche) and
Iodo-Nitrotetrazolium Violet (Sigma) were used as alkaline phosphatase
substrates. For cyp26b1 a probe was synthesized by linearizing IMAGE
clone 3722563 (Invitrogen) with SalI and transcribing with SP6.
Whole-mount immunohistochemistry, using the 3A10 antibody to detect Mauthner
cells, was performed as described (Hatta,
1992
), except a biotinylated secondary antibody and the ABC kit
(Vector Labs) were used prior to detection with FITC-Tyramide (Perkin-Elmer).
Genetic mosaic analysis was performed essentially as described
(Moens and Fritz, 1999
).
Generation of Val-monoclonal antibody, immunoblot analysis and genotyping
A GST fusion protein, where GST was fused to the full open reading frame of
Val, was used to generate monoclonal antibodies in the FHCRC Biologics
Facility. Immunoblotting was performed with hybridoma supernatant (1:4)
essentially as described (Waskiewicz et
al., 2001), except that NuPAGE sample buffer and 10% NuPAGE gels
(Invitrogen) were used. Three embryo equivalents were loaded per lane.
Genotyping of embryos with respect to val was performed as
described (Moens et al.,
1998). Genotyping for nls was performed similarly with
the primers 5'-GCTCCCACAGTAAGTTCCTGACCTA and
5'-GTTGTGGTCAGAATGGACAGACAGA, followed by digestion with excess
PstI, which cuts the mutant allele.
Morpholino injections, mRNA overexpression and pharmacological treatments
Embryos lacking both Fgf3 and Fgf8 function were generated as described
(Maves et al., 2002). mRNA for
injections was generated using the mMessage mMachine kit (Ambion) and the
following plasmids linearized with the indicated enzymes and used at the given
final concentration: pCS2+vhnf1
(Sun and Hopkins, 2001
),
NotI, 50 ng/µl; pCS2+fgf3
(Maves et al., 2002
),
NotI, 25 ng/µl; pSP64-T-caXMek
(Umbhauer et al., 1995
),
SalI, 20 ng/µl; noggin-GFP, NotI, 20 ng/µl (D.
Kimelman, personal communication). For experiments involving the injection of
more than one mRNA, total mRNA injected was normalized with eGFP
mRNA.
Pharmacological treatments of dechorionated embryos were performed in
agarose- (1.2% in embryo medium) coated dishes as follows: AGN193109
(Agarwal et al., 1996) 10 µM
in 2% DMSO in embryo medium, all-trans RA (Sigma) at given final µM
concentration in 0.1% ethanol in embryo medium.
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Results |
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To determine if RA is sufficient for the induction of vhnf1
expression, we treated embryos with all-trans RA from 8.2 hours
postfertilization (hpf) (70% epiboly) to approximately 10.7 hpf (2-3 somite
stage). Embryos treated with all-trans RA exhibited a dose-dependent expansion
of vhnf1 expression along the AP axis compared with solvent-treated
controls (Fig. 1A,B and data
not shown). To determine if vhnf1 expression requires RA signaling,
zebrafish embryos were treated with the pan-retinoic acid receptor antagonist
AGN193109 (Agarwal et al.,
1996; Johnson et al.,
1995
) beginning at 4.5 hpf, prior to the onset of gastrulation,
until approximately the 3 somite stage (11 hpf). 105 M
AGN193109, which blocks expression of val and other hindbrain markers
caudal to r4 (Linville et al.,
2004
) resulted in a severe reduction or complete loss of neural
expression of vhnf1 in all treated embryos (n=103,
Fig. 1D). Furthermore,
neckless (nls) mutant embryos, which lack Raldh2, the final enzyme in
the biosynthesis of all-trans RA (Begemann
et al., 2001
), exhibited reduced levels of vhnf1
expression (n=51/51 nls/ embryos
vs. 5/98 wild-type siblings). The effect was most striking at the end
of gastrulation, when nls embryos
(Fig. 1F) had virtually no
vhnf1 expression compared with the robust vhnf1 expression
of their wild-type siblings (Fig.
1E). vhnf1 expression in nls
embryos partially recovered by early somite stages (11 hpf), consistent with
previous studies suggesting that RA signaling is attenuated, but not fully
blocked, in zebrafish raldh2 mutants
(Grandel et al., 2002
).
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vhnf1 and Fgfs cooperate to drive val expression
Other work has shown that val expression in r5 and r6 also depends
on fgf3 and fgf8, which are strongly expressed in r4
(Maves et al., 2002;
Walshe et al., 2002
).
Expression of vhnf1 in the hindbrain does not require fgf3
and fgf8 expression in r4, and vice versa (data not shown and see
below) (Wiellette and Sive,
2003
). Therefore, we were interested in how Fgfs and
vhnf1 interact to drive val expression.
In order to determine if vhnf1 requires Fgf signaling for its ability to drive val, we overexpressed vhnf1 in wild-type (wt) embryos and embryos lacking Fgf3 and Fgf8 function. In wt embryos, overexpression of vhnf1 by mRNA injection expanded the val expression domain anteriorly (Fig. 2A,B) to include the r2-r4 territories. By contrast, overexpression of vhnf1 in embryos lacking Fgf3 and Fgf8 function did not drive val expression in either its endogenous domain of r5 and r6 or ectopically in more anterior rhombomeres (Fig. 2C). These data demonstrate that activation of val by Vhnf1 strictly depends on Fgf signaling.
|
One of the key downstream effectors of Fgf signaling is the Ras-Map kinase
(MapK) signaling cascade (Powers et al.,
2000). To determine if Fgf signaling through MapK synergizes with
vhnf1 to drive val, we coexpressed vhnf1 with a
constitutively active MapK/ERK kinase (caMek) mRNA
(Umbhauer et al., 1995
). caMek
alone did not drive val expression
(Fig. 2J) but cooperated with
vhnf1 to drive val expression in a manner similar to that of
Fgf (Fig. 2K).
The normal expression of val in r5 and r6 is dependent on a
positive autoregulatory loop (Giudicelli
et al., 2003; Moens et al.,
1998
). In order to determine if the vhnf1-fgf3
interaction we observed is dependent on this positive autoregulatory loop, we
repeated the vhnf1-fgf3 overexpression experiments in embryos from a
val+/ intercross. We found that strong expression
of val at 8.5 or 12 hpf correlated with wt or heterozygous genotype
(94%, n=98, Fig. 2L),
while nearly all embryos with little to no val expression were mutant
(88%, n=51, 2M). These data suggest that val autoregulation
is required for vhnf1 and fgfs to drive robust val
expression.
Vhnf1 controls some aspects of rhombomere identity independently of Val
The above data and that of others demonstrate that RA activates
vhnf1, which in cooperation with Fgfs is both necessary and
sufficient to drive expression of val in r5 and r6. To determine
whether vhnf1 performs all its functions in hindbrain development
through its regulation of val, or whether the functions of
val and vhnf1 are in part separable, we carefully compared
molecular and neuroanatomical markers of rhombomere identity in
val and vhnf1 embryos.
If the principal role of vhnf1 in specifying hindbrain fates is the
initiation of val expression, val and
vhnf1 embryos should exhibit identical
phenotypes.
Both val and vhnf1 mutant embryos failed to develop a
recognizable r5-r6 territory. This is exemplified by the loss of r5
krox20 expression (Fig.
3A-C), of hox3 gene expression and of r5-6 specific
abducens motoneurons (data not shown) in both mutants. However, there are
subtle differences in the two mutant phenotypes. For example, in val
mutants the remnant of r5 krox20 expression is always in the
dorsalmost hindbrain (Fig. 3B),
while vhnf1 mutants often have fewer krox20-positive cells
than do val mutants and many of the cells are located ventrally
(Fig. 3C). Furthermore, the
r5-6 region of val mutants is about the length of a single rhombomere
(Moens et al., 1996), while
that of vhnf1 mutants is the same as in wild-type embryos (data not
shown). This is unexpected if vhnf1 performs all its functions
through val.
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While some r4 markers behave differently in vhnf1 and val
mutants, other r4 markers are unaffected in either mutant. cyp26b1,
which encodes a retinoic acid-degrading enzyme, is expressed in r4 beginning
at tailbud stage and expands to include r3 by 12 hpf (R.E.H. and C.B.M.,
unpublished). fgf8 is expressed in a domain anterior to the r4-5
boundary at 12 hpf (Reifers et al.,
1998). Surprisingly, neither marker was expanded posterior to r4
in either val or vhnf1 mutants at a stage when
hoxb1a was strongly expanded in vhnf1 mutants
(Fig. 3S-X).
Wiellette and Sive (Wiellette and Sive,
2003) reported that the Mauthner cell, a large identified
reticulospinal neuron characteristic of r4, is duplicated in more posterior
segments in vhnf1 mutants. Our finding that the transformation to r4
identity in vhnf1 mutants was incomplete based on marker gene
expression led us to reexamine Mauthner cell specification. We found that only
58% of mutants had one or more supernumerary Mauthner cells, consistent with
an incomplete transformation (n=19,
Fig. 3AA). val mutants
never had supernumerary Mauthner cells (n=19,
Fig. 3Z), consistent with only
a very weak anterior transformation.
Taken together, our marker analysis demonstrates that vhnf1 function in hindbrain patterning is only partially executed through its activation of val. Both vhnf1 and val are required for upregulation of r5 and r6 markers, but vhnf1 functions largely independently of val to repress some aspects of r4 identity. However, even in the absence of vhnf1 function the r5-6 territory appears to be specified properly initially, including transient restriction of r4-specific genes. vhnf1, but not val, is strongly required to maintain repression of some, but not all, r4 markers, and the partial transformation of the r5-6 region of vhnf1 mutants to r4 identity correlates with a variable gain of r4-specific neurons.
vhnf1 and val mutant cells behave equivalently in genetic mosaic analysis
Individual rhombomeres have specific cellular surface characteristics and
the involvement of individual genes in the acquisition of this aspect of
rhombomere identity can be determined by genetic mosaic analysis. Previous
work has shown that val is required cell autonomously for the
acquisition of r5 and r6 identity (Moens
et al., 1996). Similarly, vhnf1 mutant cells were
excluded from r5 and r6 of wild-type hosts
(Fig. 4B, n=20,
compare with control in Fig.
4A), only occasionally contributing to the r5-6 boundary of the
host hindbrain. Conversely, wild-type cells typically formed compact clusters
within the presumptive r5-6 of vhnf1 hosts
(arrowheads in Fig. 4C,
n=29) and the clusters of cells located in the presumptive r5
expressed krox20. These dense clusters were unilateral, consistent
with a failure of the transplanted wild-type cells to make a characteristic
division that requires single cells to insert themselves across the midline
(Geldmacher-Voss et al., 2003
;
Kimmel et al., 1994
). These
data demonstrate that vhnf1 is required cell autonomously for cells
to acquire r5 and r6 identities.
|
Cell sorting in the hindbrain is probably controlled by repulsive
interactions between Ephs and Ephrins
(Cooke et al., 2001;
Mellitzer et al., 1999
;
Xu et al., 1999
). Therefore,
we determined whether the cell-surface properties of
val and vhnf1 cells
observed in our mosaic analysis were reflected in Eph and
ephrin expression patterns. In spite of marked differences in
hoxb1a and fgf3 expression in the two mutants, Eph
and ephrin expression were very similar. In both
vhnf1 and val embryos,
expression of EphB4a in r5 and r6 and of EphA4 in r5 was
greatly reduced (Fig. 4F-H and
data not shown); this reflects changes in krox20 and hox3
expression, which were also similar in the two mutants. By contrast,
ephrin-B2a, which is normally not expressed in r5 or r6, was expanded
in both val and vhnf1
embryos (Fig. 4I-K)
(Cooke et al., 2001
). The
similar cell-sorting behavior of vhnf1 and
val cells in genetic mosaics would not have been
predicted by the examination of Hox expression alone. These results
demonstrate that rhombomere-specific neuronal identity as determined by a
`hox code' can be unlinked from rhombomere-specific cell-surface
properties as determined by an `Eph-ephrin code'.
Ectopically expressed vhnf1 represses hoxb1a and r4-ephrin-B2a in a val dependent manner
The expansion of hoxb1a and ephrin-B2a in vhnf1
mutants suggests that vhnf1 normally represses the expression of
these two genes. Since ephrin-B2a expression is significantly
expanded in val embryos while hoxb1a is
not, we hypothesized that vhnf1 repression of ephrin-B2a
requires val while vhnf1 repression of hoxb1a does
not. In order to test this hypothesis, we overexpressed vhnf1 in
embryos from an intercross of heterozygous val fish and then assessed
the expression of hoxb1a and ephrin-B2a by in-situ
hybridization.
Consistent with vhnf1 playing a role in the repression of hoxb1a and ephrin-B2a, we observed that wild-type embryos injected with vhnf1 mRNA showed repression of hoxb1a (88%, n=114) and ephrin-B2a (88%, n=91) in r4 at about 5-9 somites (11.5-13 hpf). Many of these embryos had nearly a complete loss of the r4 hoxb1a or ephrin-B2a domain, with only a few hoxb1a (Fig. 5B) or ephrin-B2a (Fig. 5F) positive cells remaining between two-fused krox20 stripes. However, essentially no homozygous val embryos showed repression of hoxb1a (6%, n=51, Fig. 5D) or ephrin-B2a (0%, n=33, Fig. 5H) in r4 following overexpression of vhnf1.
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Discussion |
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It remains unclear whether the activation of vhnf1 by RA is
direct, as has been determined for some Hox genes
(Dupe et al., 1997;
Gould et al., 1998
;
Marshall et al., 1994
), or if
it acts through a more indirect mechanism. Members of the steroid nuclear
receptor superfamily, including RARs, can bind a DR1 motif upstream of murine
Vhnf1, and the DR1 is required for full reporter activity in cultured
cells, but it is unclear if this element mediates retinoic acid responsiveness
(Power and Cereghini, 1996
).
Pbx proteins, which function as DNA-binding partners for Hox proteins, are
also required for normal hindbrain expression of vhnf1
(Waskiewicz et al., 2002
).
Since Pbx proteins are not known to be required for RA synthesis or to be
regulated by RA, their requirement for vhnf1 expression is likely to
be either independent or downstream of RA signaling. A careful analysis of the
vhnf1 promoter will help elucidate which proteins directly regulate
vhnf1 in the hindbrain.
vhnf1 and Fgfs synergize to drive val expression
We have shown that vhnf1 and Fgfs cooperate to drive val
expression and specify r5 and r6 fates. Using Fgf-coated beads, Wiellette and
Sive (Wiellette and Sive,
2003) demonstrated a similar synergy between Fgf and
vhnf1. Our results support and extend their findings, by showing that
unlike RA signals, Fgf signals are strictly required for vhnf1 to
induce val expression, since vhnf1 cannot drive val
expression in an embryo lacking fgf3 and fgf8. Furthermore,
we demonstrate that Fgf signaling through the MapK pathway is sufficient to
cooperate with vhnf1 in inducing val expression, and that
robust upregulation of val by Vhnf1 and Fgfs requires a positive
autoregulatory loop that is dependent on Val function.
The control of val expression by vhnf1 is probably
conserved across vertebrates. vhnf1 is expressed in the hindbrain of
mouse embryos in a similar domain to that in zebrafish, but its requirement
for hindbrain development has not been determined because homozygous mutant
mice have defects in visceral endoderm development and die prior to
gastrulation (Coffinier et al.,
1999). Inactivation of vhnf1 in the embryonic tissues or
CNS alone will be required to test the requirement for vhnf1 in
mammalian hindbrain patterning.
At this point it is unclear whether vhnf1 acts directly or
indirectly to regulate expression of val, and little is known about
the direct regulation of the val locus. The classical
kreisler mutation, an inversion approximately 30 kb upstream of the
transcriptional start of Kr/MafB, disrupts a regulatory element that
is specifically required for expression in r5 and r6 but not sites outside the
hindbrain (Cordes and Barsh,
1994; Eichmann et al.,
1997
). This and other data
(Hamada et al., 2003
) suggest
that elements controlling the hindbrain expression of val/mafB/Kr are
rather distant from the gene. We have identified a consensus Hnf1-binding site
(CTGTTAACATAACA) within a highly conserved island of homology (85% identity
over 500 base pairs) approximately 22.5 kb upstream of the
MafB/Kr gene of humans and mice (data not shown). However,
we have as yet been unable to identify a corresponding island in the available
genomic sequence from either fugu or zebrafish. Further analysis is
necessary to determine if this potential Hnf1 binding site has any role in
regulating hindbrain expression of val/mafB/Kr.
Although our data and that of Wiellette and Sive
(Wiellette and Sive, 2003)
show that Fgfs synergize with Vhnf1 to drive val expression, the
mechanism underlying this effect is unclear. Our data suggest that Fgf
signaling through the MapK cascade promotes Vhnf1 protein activity. We
considered the possibility that MapKs may directly regulate Vhnf1 by
phosphorylation. However, Vhnf1 is a poor substrate for Erk2 in an in-vitro
assay and has only marginal consensus MapK phosphorylation sites (data not
shown). More indirect mechanisms, in which MapK-signaling activates other
proteins or expression of intermediate target genes, remain to be
investigated.
Different aspects of rhombomere identity are regulated independently by Val and Vhnf1
The specification of r5 and r6 identities requires both the activation of
r5- and r6-specific genes and the repression of r4-specific genes. Our data
demonstrate that while vhnf1 and val probably function in a
linear pathway to activate r5- and r6-specific genes, the repression of
r4-specific genes is more complex. Vhnf1 functions through Val to repress
ephrin-B2a and thus repress r4-specific cell-surface properties, but
it does not require Val to repress hoxb1a and thus repress
r4-specific neuronal differentiation. As a result, vhnf1 and
val mutants have different `hox codes' and patterns of
neuronal differentiation, but have similar `Eph-ephrin codes' and
cell behaviors in genetic mosaics. These results show that different aspects
of segment identity, in this case neuronal phenotype and cell-surface
character, can be regulated independently.
vhnf1 and val participate in a multistep process to repress the r4 `hox code'
Wiellette and Sive (Wiellette and Sive,
2003) propose that a sweep of r4 identity from posterior to
the r3/4 boundary is subsequently restricted to r4 by the expression of
vhnf1. However, the expansion of r4 fates in vhnf1 mutants
is not complete, suggesting that multiple factors are required for the
restriction of r4 fates.
Our analysis of r4 marker expression shows that hoxb1a expression
is transiently downregulated posterior to r4 in vhnf1 mutants, as in
wt embryos. Thus, an unknown factor functions to repress the earliest
hoxb1a expression in the presumptive r5/6 territory. After the onset
of its expression, vhnf1 rapidly becomes the primary repressor of
hoxb1a expression. However, vhnf1 does not repress all
r4-specific gene expression as predicted by Wiellette and Sive
(Wiellette and Sive, 2003),
since fgf8 and cyp26b1 are restricted anterior to the r4/5
boundary even in vhnf1 mutants. As a result, the expanded `r4'
territory in vhnf1 mutants has different molecular identity from r4
proper. Consistent with this incomplete transformation, the duplication of
r4-specific Mauthner cells in vhnf1 mutants is not fully penetrant.
Together, these results show that r4 is distinguished from the more posterior
hindbrain by more than simply the expression of vhnf1 posterior to
the r4-5 boundary.
Once the repression of hoxb1a is strongly established by Vhnf1,
the expression of vhnf1 recedes from the hindbrain. This coincides
with the period that hoxb1a expands slightly in
val embryos
(Prince et al., 1998),
suggesting that Val is required at later stages to maintain repression of
hoxb1a in r5 and r6. Val may function to repress hoxb1a by
activating hox3 genes, which have been shown to be required for the
maintained repression of hoxb1 in the mouse
(Gaufo et al., 2003
).
val is required for the repression of the r4 `Eph-ephrin' code and the establishment of r5-6 cell adhesive properties
Wiellette and Sive (Wiellette and Sive,
2003) suggested that vhnf1 may function non-autonomously
through an unknown signal to specify the most anterior r5 fates, because they
did not observe vhnf1 expression extending to the r4-5 boundary. We
have seen that the domain of vhnf1 expression does include the entire
r5 domain of krox20 expression
(Fig. 1A). Furthermore, our
mosaic analysis demonstrates that vhnf1 is required cell-autonomously
for the acquisition of r5 and r6 fates
(Fig. 4B,C), since
vhnf1 cells are excluded from r5 and r6 of
wild-type hosts.
In the absence of either val or vhnf1, cells in the r5-6
region acquire the same cell-surface properties as determined by reciprocally
transplanting cells between the two mutants
(Fig. 4D,E). This is in direct
contrast to the distinct molecular phenotypes of the two mutants, including
hoxb1a expression, but correlates with their similar patterns of
Eph and ephrin expression. Cooke and colleagues
(Cooke et al., 2001)
demonstrated that cell sorting in val mosaics was attributable to
repulsive signals between val ephrin-expressing
cells and wt Eph-expressing cells; the same mechanism probably
explains the cell sorting we observed in vhnf1 mosaics. The similar
effects on Eph and ephrin expression in val and
vhnf1 mutants, and our observation that vhnf1 requires
val to repress ephrin-B2a when it is overexpressed, suggest
that vhnf1 functions through val to specify the cell-surface
character of the r5-6 region, including repression of r4-specific adhesive
character (i.e. ephrin-B2a).
Together, our data support a multistep model for the initial restriction of
r4 identity and the specification of r5-6 development
(Fig. 6). r4 identity is
initially restricted by the repression of hoxb1a in the presumptive
r5-6 region by an unknown, vhnf1-independent, mechanism.
vhnf1 expression is activated by RA up to the r4-5 boundary and
strictly reinforces the restriction of hoxb1a to r4, thereby limiting
the expression of fgf3 and development of r4-specific Mauthner
neurons. Vhnf1 also cooperates with Fgfs expressed in r4 to activate the
expression of val and the r5-6 program of development. val
subsequently drives the expression of r5-6 specific Hox genes and the
development of r5-6-specific neurons. Although val is not strongly
required for the repression of hoxb1a expression, it is required for
the repression of r4-like cell-surface characteristics that drive cell sorting
as mediated by Eph and ephrin expression. The different
requirements for vhnf1 and val in the specification of
`hox code' and `Eph-ephrin code' demonstrate that the
mechanisms that specify segmental neuronal identity and differential
cell-surface characteristics between rhombomeres in the hindbrain can be
independently regulated. Previous work showing that Eph and ephrin expression
are regulated by krox20 and val/mafB/Kr rather than by Hox
genes (Cooke et al., 2001;
Theil et al., 1998
) had
predicted that cell-surface characteristics could be regulated independently
from other aspects of segment identity. However, due to cross-regulation
between krox20, val and Hox genes, Hox expression and
Eph-ephrin expression are generally coupled, so this prediction has
not been tested. Our discovery of an instance in which Hox expression and
Eph-ephrin expression are unlinked has allowed us to show directly
that neuronal identity corresponds with `hox code', while cell
sorting behaviors correspond with `Eph-ephrin' code.
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ACKNOWLEDGMENTS |
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Footnotes |
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