1 Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge,
MA 02142, USA
2 Massachusetts Institute of Technology, Cambridge MA, USA
* Author for correspondence (e-mail: sive{at}wi.mit.edu)
Accepted 1 May 2003
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SUMMARY |
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Key words: Vhnf1, Fibroblast growth factor, Fgf3, Fgf8, Zebrafish, Hindbrain, Valentino, Krox20, Rhombomere, Neural patterning
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INTRODUCTION |
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By late gastrula stages, cells that will contribute to the hindbrain are
already committed to that fate (Woo and
Fraser, 1998). At this time, a broad region of the presumptive
posterior neurectoderm is distinguished by the expression of genes including
hoxa1, hoxb1 and meis3
(Alexandre et al., 1996
;
Kolm and Sive, 1995
;
Murphy and Hill, 1991
;
Prince et al., 1998
;
Sagerstrom et al., 2001
;
Salzberg et al., 1999
).
Further, because the anterior boundary of hoxA1 gene expression
probably lies at the future r3-r4 break and only the posterior hindbrain
tissue up to the r3-r4 boundary is dependent on retinoic acid (RA), the
presumptive caudal hindbrain (r4-r8) appears to compose an early, distinct
domain (Dupe and Lumsden, 2001
;
Gavalas and Krumlauf,
2000
).
The posterior hindbrain is subsequently subdivided, as indicated by
restricted gene expression, including krox20 (egr2
Zebrafish Information Network) in r3 and r5, and valentino
(val)/kreisler/mafB in r5 and r6. Both
krox20 and val functions are required for the correct
expression of some of the rhombomere-specific Hox genes
(Frohman et al., 1993;
Giudicelli et al., 2003
;
Manzanares et al., 1999
;
Prince et al., 1998
;
Seitanidou et al., 1997
). In
turn, Hox gene expression domains delineate presumptive rhombomeres, and Hox
gene function is required for the development of neurons and other cells
produced within each rhombomere (Lumsden
and Krumlauf, 1996
; Moens and
Prince, 2002
; Trainor and
Krumlauf, 2001
). For instance, hoxb1 is expressed in
future rhombomere 4 (r4) and is sufficient to provide ectopic r4 neuronal
morphology (Bell et al., 1999
;
Vlachakis et al., 2001
). In
combination with hoxa1, hoxb1 is required for normal development of
presumptive r4 in mice and zebrafish
(Gavalas et al., 1998
;
McClintock et al., 2002
;
Rossel and Capecchi, 1999
;
Studer et al., 1998
).
Similarly, the Hox paralog group 3 genes are expressed in r5 and r6 and are
required for formation of specific neurons and mesenchymal neural
crest-derived structures (Manley and
Capecchi, 1997
).
Recent findings identify some additional factors required for posterior
hindbrain segmentation. In the chick, fibroblast growth factor (Fgf) signals
have been shown to be sufficient for ectopic induction of krox20 and
mafB/kreisler in caudal hindbrain neuroepithelium and neural
crest, and drug-based inhibition of Fgf signaling results in inhibition of
krox20 and mafB/kreisler within their normal
expression domains (Marin and Charnay,
2000). In the zebrafish, Fgf signals emanating from the anterior
hindbrain are required to initiate expression of posterior hindbrain gene
expression, in particular val, krox20 and hoxb3. Loss of
both fgf3 and fgf8 functions together results in a loss of
r5 and r6 identity (Maves et al.,
2002
; Walshe et al.,
2002
). Loss of function of the gene variant hepatocyte nuclear
factor 1 (vhnf1; tcf2 Zebrafish Information
Network) in the zebrafish results in small ears and loss of val and
krox20 (r5) expression (Sun and
Hopkins, 2001
). Although vhnf1 knockout mice have been
made, the role of vhnf1 in murine hindbrain development has not been
studied (Barbacci et al., 1999
;
Coffinier et al., 1999
).
It remains to be defined how the broad domain of gastrula stage posterior neuroectodermal gene expression is subdivided into individual rhombomeric domains. Partially explaining this, we show here that vhnf1 is expressed in a broad domain during gastrulation and that it is required for differentiation of caudal hindbrain rhombomeres by two distinct mechanisms. Through an obligate synergy with Fgf signals, vhnf1 promotes expression of val, thereby promoting r5 and r6 identity. In addition, vhnf1 represses hoxb1a expression independently of Fgf function, thereby limiting r4 identity to the appropriate narrow domain.
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MATERIALS AND METHODS |
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RNA injections
pCS2+ plasmids with cDNA containing vhnf1
(Sun and Hopkins, 2001),
val (Moens et al.,
1998
) or lacZ were linearized and transcribed using the
mMessage mMachine kit (Ambion). Capped mRNA concentration was measured and
RNAs were injected in the following final amounts: vhnf1, 25 pg;
lacZ, 25 pg; val, 5 pg. ß-Galactosidase (ß-gal)
was visualized after fixation of embryos overnight in BT fix
(Westerfield, 1995
) at 4°C
by washing in PBT and then staining in ß-gal stain buffer (1x PBS,
4 mM MgCl2, 3 mM K4[Fe(CN)6], 3 mM
K3[Fe(CN)6]) + 0.2% X-gal at room temperature. Embryos
were analysed first by ß-gal stain, followed by dechorionation,
dehydration in methanol overnight and then the standard in situ method (see
below).
Morpholino oligo injections
To knock down the functions of the fgf3 and fgf8 genes,
morpholino oligomers (MOs) targeted to the translation start sites
(Raible and Brand, 2001) were
injected into 1-2 cell embryos. The final concentrations used were: 2.5 ng of
each MO (Fig. 3C,D) or 1 ng of
each MO (Fig. 5). In each case,
two controls were performed in which a double concentration of one oligo or
the other was used and, in each case, the strongest effect was observed by
injection of the combination of MOs directed against both fgf
transcripts. For analysis of earlier staged embryos
(Fig. 3E,F), 0.8 ng of each MO
was injected, with an unrelated negative control MO used to make 1.6 ng total
MO in the injections of either fgf3MO or fgf8MO alone.
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|
Reticulospinal neuron labeling
Reticulospinal neurons were labeled in embryos fixed at 48 hours of
development, using 1:50 dilution of the primary antibody RMO44
(anti-Neurofilament; Zymed Laboratories #13-0500), as described
(Waskiewicz et al., 2001).
Localization of RMO44 was visualized using a 1:50 dilution of
FITC-
-mouse (Zymed). The brains were partially dissected and mounted
for visualization by confocal microscopy.
RNA injection with bead implantation
Beads were prepared and coated in mouse Fgf8b protein (R&D Systems) [or
bovine serrum albumen (BSA)] as described in Reifers et al. (Reifers et al.,
2000). vhnf1 or val mRNA was injected at the 2-cell stage
and the embryos were allowed to grow until the shield stage. Injected embryos
were placed in the lid of a small Petri dish lightly coated with 3%
methylcellulose and covered with normal Ringer's solution
(Westerfield, 1995). A needle
prepared as for injection was used to tear a small hole in the ectoderm and
sharp forceps were then used to pick up a single bead and push it into the
incision. The needle was then used to push the bead farther under the
ectoderm. When all embryos on a dish lid were treated, the lid was placed in a
standard Petri plate, which was then flooded with Embryo Medium
(Westerfield, 1995
). Embryos
were left untouched until the appropriate stage for fixation, at which point
they were gently removed from the methylcellulose and transferred for
fixation, ß-gal staining and in situ hybridization (as above).
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RESULTS |
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Morphological defects are consistent with the observed pattern formation
defects. The reticulospinal neurons that develop in the hindbrain are visible
by 48 hours of development, including the large Mauthner neurons, which are
derived from r4 (Fig. 1K). vhnf1wi408 mutants develop excess Mauthner neurons in
parallel with loss of r5+r6-derived neurons
(Fig. 1L). Although the normal
r4-derived Mauthner neuron pair is always present in the correct location, the
extra Mauthner neurons in vhnf1wi408 mutants are routinely
observed both within the normal locale for Mauthner neurons and in more
posterior locations. In addition to the neuron identity changes, the otic
vesicle is small and round (data not shown)
(Sun and Hopkins, 2001).
Together with published data, the gene misexpression and morphological defect
data are consistent with a transformation of rhombomere identity from r5+r6 to
r4 during late gastrula and tailbud stages.
vhnf1 is expressed during gastrulation in the caudal
hindbrain
It has been shown that vhnf1 expression is present in the
hindbrain by tailbud stage and that the anterior boundary of vhnf1
expression lies within the presumptive r5 domain by the four-somite stage
(Sun and Hopkins, 2001).
Because vhnf1 regulates val expression before tailbud stage,
we wanted to generate an accurate description of the timing and initial
localization of vhnf1 expression. An excellent marker of relative
position in the gastrulating embryo is hoxb1b expression, which
appears at early gastrula stages, and which has a sharp anterior border
(zebrafish hoxb1b is the same as mouse hoxa1)
(Alexandre et al., 1996
). It is
not clear what region of the hindbrain will be derived from this early domain
of Hoxb1a expression, but it is likely to map at or near the future r3-r4
boundary. In situ hybridization of hoxb1b and vhnf1 shows
that localized hindbrain expression of vhnf1 begins after (not shown)
and with a more posterior boundary than that of hoxb1b
(Fig. 2A). Co-localization of
fgf8 and vhnf1 transcripts shows that vhnf1
expression begins before that of fgf8 in the hindbrain (not shown)
and that fgf8 expression is more anterior than that of vhnf1
(Fig. 2B). There is a
persistent gap of two or three cells between these fgf8 and
vhnf1 expression domains. fgf8 expression abuts
hoxb1b expression throughout late gastrula and tailbud stages
(Fig. 2C).
|
Fgf signals are epistatic to vhnf1 function
To understand better the molecular interactions that allow vhnf1
to generate r5+r6 identity, we considered other defects that cause a similar
phenotype. fgf3 and fgf8 are expressed in the presumptive
anterior hindbrain (Fig. 2G),
and their partially redundant functions can be ablated by injection of MOs
(Raible and Brand, 2001). Loss
of Fgf signaling in the hindbrain results in loss of r5+r6 identity
(Maves et al., 2002
;
Walshe et al., 2002
). However,
although loss of vhnf1 function results in expansion of r4 identity
(Fig. 3A,B), loss of Fgf
signals gives no expansion of hoxb1a
(Fig. 3C) (Maves et al., 2002
;
Walshe et al., 2002
). In this
fgf loss-of-function background, hoxB4 expression does not
expand to the anterior, suggesting that there is no posteriorization of the
tissue (Fig. 3D).
To test whether an epistatic relationship exists between vhnf1 and Fgf signals, synthetic double mutants were made. Injection of fgf3 + fgf8 MOs into embryos derived from vhnf1wi408 carrier parents produced embryos all of the same phenotype, with hoxb1a expression limited to the r4 domain and hoxB4 expression limited to the r6-r7 boundary (Fig. 3C,D). Because the loss of the combination of vhnf1 function and Fgf signals gives the same phenotype as loss of Fgf signals alone, Fgf signaling appears to be epistatic to vhnf1 function in hindbrain pattern formation.
To determine whether the epistasis between vhnf1 and Fgfs results from regulation of transcript levels, expression of each gene was tested in the other mutant background. Knockdown of Fgf signaling by injection of a mix of fgf3 + fgf8 MOs results in no change in the anterior-posterior pattern of vhnf1 expression (Fig. 3E). The effectiveness of the MOs to knock down Fgf function in the posterior hindbrain was monitored by loss of r5 krox20 expression in sibling embryos (Fig. 3F). Embryos generated from vhnf1wi408 heterozygous parents all show the same pattern of fgf8 and fgf3 transcript expression at the one-somite stage, and so vhnf1 function is not required for normal fgf8 or fgf3 expression at this stage (Fig. 3G; not shown). Thus, Fgf signals function epistatically to vhnf1 to generate rhombomere identity in the posterior hindbrain, and this epistatic relationship is not based on regulation of RNA levels during gastrulation (Fig. 3H).
vhnf1 function and Fgf signals synergize to regulate caudal
hindbrain genes
Although both Fgf signals and vhnf1 function are required to
specify r5+r6, neither factor alone is sufficient. Ectopic vhnf1
induces r5 identity only in the r4 domain, and ectopic Fgf has a limited
ability to induce r5+r6 identity at late somitogenesis and primarily within
the caudal hindbrain (Maves et al.,
2002; Sun and Hopkins,
2001
). Therefore, the combination of these two factors was tested
for an enhanced ability to induce r5+r6 identity.
vhnf1 RNA was injected into one cell at the two-cell stage. Injected embryos were grown to shield stage, at which point an Fgf8-coated bead was inserted. Embryos were then grown to approximately the three-somite stage and fixed (Fig. 4A). Injection of vhnf1 RNA and implantation of an Fgf8-coated bead results in strong induction of krox20 expression limited to the cells expressing vhnf1 and lying close to the Fgf8 bead (18/20 embryos). This effect is particularly strong throughout the neural plate (Fig. 4B), and is also robust in non-neural ectoderm (Fig. 4C). Embryos in which a BSA-coated bead was implanted near cells overexpressing vhnf1 show no induction of krox20 expression (6/6 embryos) (Fig. 4D). The Fgf8 bead alone does not induce any krox20 expression (10/10 embryos) (Fig. 4E), unless the bead lies adjacent to the normal domain of krox20 expression (not shown). To determine whether induced krox20 expression has r5-specific identity, induction of val expression was also examined and the combination of vhnf1 expression with the Fgf8 bead was found to result in induction of val expression (5/5 embryos) (Fig. 4F). It appears that the combination of vhnf1 and Fgf8 is sufficient for induction of early r5+r6 gene expression.
|
To determine whether Fgf8 and val can synergize in a manner similar to Fgf8 and vhnf1, val RNA was injected and Fgf8-coated beads were added to the embryos. In this case, no induction of krox20 expression is observed (15/19 embryos) (Fig. 4H), although weak krox20 induction is observed in a few cases in which the bead is located close to the endogenous krox20 domain (4/19 embryos). Thus, Fgf8+val is insufficient to activate ectopic krox20 expression, suggesting that vhnf1 is specifically required to make hindbrain cells competent to respond to Fgf8 signals. In addition, this result shows that val RNA alone is insufficient to induce ectopic krox20 expression (Fig. 4H). Therefore, it is likely that additional factors induced by Fgf+vhnf1 are required in collaboration with val to activate krox20 expression.
To test whether vhnf1+Fgf8 can induce caudal hindbrain identity other than r5+r6, the expression of hoxb1a, a presumptive r4 marker, was analysed. hoxb1a expression is partially repressed in the region where vhnf1 expression overlaps endogenous hoxb1a expression (10/10 embryos) and is not ectopically induced in regions where the Fgf8 bead lies close to the vhnf1 expression (4/4 embryos) (Fig. 4I). Fgf+vhnf1 is therefore insufficient to induce ectopic r4 identity.
Fgf8 can induce mesoderm identity
(Griffin et al., 1998;
Rodaway et al., 1999
) and
mesoderm is a source of neural posteriorizing signals including Fgfs
(Koshida et al., 1998
;
Kudoh et al., 2002
;
Woo and Fraser, 1997
). The
expression of no tail (ntl) was examined to determine
whether mesoderm was induced by Fgf8 under these experimental conditions and
could mediate the synergy between vhnf1 and Fgf8. However, no ectopic
ntl expression is observed in most embryos (13/15)
(Fig. 4J), although, when the
bead lies deep under the epiblast and close to the prechordal plate, ectopic
ntl is observed close to the beads in deep tissue regions (2/15
embryos). Therefore, the co-operativity of Fgf signals and vhnf1
function appears to occur within the ectoderm. In summary, Fgf signals
synergize with vhnf1 to activate expression of r5+r6-specific genes,
including the activation of val and krox20 expression
(Fig. 4K).
vhnf1 represses hoxb1a independent of Fgf signals
and val function
The expansion of hoxb1a in vhnf1wi408 mutants
(Fig. 1B), paired with the
suppression of hoxb1a by injection of vhnf1 RNA
(Fig. 4I), suggests that one of
the ways in which vhnf1 functions is to repress r4-specific
hoxb1a expression. Therefore, we further examined hoxb1a
expression in response to ectopic vhnf1. Expression of
hoxb1a at 90-100% epiboly is normal when vhnf1 is
overexpressed (Fig. 5A).
However, by the six-somite stage, expression of hoxb1a is completely
repressed by overexpression of vhnf1, and krox20 expression
appears in a single broad band (32/32 embryos)
(Fig. 5B). Overexpression of
lacZ alone has no effect on hoxb1a or krox20
expression (17/17 embryos) (Fig.
5C). Thus, ectopic vhnf1 is able to repress
hoxb1a expression in presumptive r4.
To test whether the repression of hoxb1a by vhnf1 occurs through val function, the ability of vhnf1 to repress hoxb1a in a val mutant background was tested. Ectopic vhnf1 is able to repress hoxb1a expression in this background (10/10 embryos) (Fig. 5D). val mutant embryos injected with lacZ do not show repression of hoxb1a (7/7 embryos) (data not shown). No krox20 expression remains after vhnf1 overexpression in val mutants, indicating that ectopic vhnf1 represses r3-specific krox20 as well as r4-specific hoxb1a. Therefore, it appears that ectopic vhnf1 transforms both presumptive r3 and r4 towards the r5 identity autonomously of val function.
To test whether the repression of hoxb1a by vhnf1
requires the co-function of Fgf signals, the ability of ectopic vhnf1
to repress hoxb1a in a fgf-compromised background was
tested. Embryos were injected with MOs directed against fgf3 and
fgf8 along with the vhnf1 RNA. Ectopic vhnf1
represses hoxb1a expression in the absence of Fgf signals (22/22
embryos) (Fig. 5E) but
co-injection of lacZ with the fgf MOs does not reduce the
extent of hoxb1a expression (59/59 embryos)
(Fig. 5F). These data imply
that vhnf1 functions independently of Fgf signals to repress
hoxb1a expression. Because knockdown of fgfs based on the
MOs used in this experiment does not lead to complete repression of r5
krox20 expression, it is interesting that there is no remaining
krox20 expression in the tissue injected with vhnf1 RNA and
anti-fgf MOs. This might reflect a synergistic effect that results
from repression of fgf8 expression by vhnf1
(Fig. 5G,H), which might give a
stronger loss of fgf functions and therefore a complete repression of
r5 krox20 expression. fgf8 is expressed throughout
presumptive anterior hindbrain tissue at tailbud stage
(Reifers et al., 1998), and
vhnf1 appears to be sufficient to repress anterior hindbrain identity
during gastrulation (Fig.
5G).
val can partially rescue loss of vhnf1
function
The expression of multiple genes is dependent on vhnf1 function,
including val, krox20 and hoxb3. Of these, val is
the earliest expressed, and loss of val function also results in loss
of krox20 in presumptive r5 and loss of hoxb3 expression
(Moens et al., 1996;
Prince et al., 1998
). To test
whether vhnf1 is independently required for regulation of
krox20 and hoxb3, val RNA was injected into
vhnf1wi408 mutant embryos and expression of target genes
was analyzed. Expression of krox20 is unaffected in wild-type embryos
by overexpression of val (Fig.
6A). However, injection of val RNA is sufficient to
rescue r5 krox20 expression in vhnf1wi408 mutant
embryos in the injected side of the embryo (7/8 embryos) and it is striking
that krox20 expression induced by val RNA is limited to the
presumptive r5 domain (Fig.
6B). lacZ RNA alone has no ability to rescue
krox20 expression (Fig.
6C).
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DISCUSSION |
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Subdivision of the caudal hindbrain domain
The data presented here define the mechanism by which the initially broad
expression domains of the future hindbrain are subdivided
(Fig. 7). Soon after
hoxb1b expression appears with an anterior limit near the future
r3-r4 boundary, vhnf1 expression begins, with a more posterior limit
of expression, probably within the future r5 region. It is not clear how
vhnf1 expression is initiated or what limits its anterior boundary.
Expression of vhnf1 is sensitive to retinoic acid (RA) (E. Wiellette
and H. Sive, unpublished) and RA receptor binding sites have been identified
in the mouse vhnf1 promoter
(Power and Cereghini, 1996),
so it is possible that RA directly regulates vhnf1 transcription.
However, it is also possible that the observed RA sensitivity is mediated by
hoxb1b function; when Hox gene function is removed from zebrafish by
making embryos devoid of pbx gene functions, vhnf1
transcription is not initiated (Waskiewicz
et al., 2002
).
|
vhnf1 function is required for activation of val and
krox20 expression. However, the anterior boundary of vhnf1
expression lies posterior to the anterior boundary of krox20 r5
expression, which is comparable to that of val
(Moens et al., 1998). Because
vhnf1 encodes a putative transcription factor, it seems likely that
Vhnf1 protein is not acting directly to regulate the transcription of
val or krox20 but might rather be regulating transcription
of an extracellular signal, which works at a distance of one or two cell
diameters to control the expression of val and krox20.
Also, at the end of gastrulation, hoxb1a expression appears in a
broad domain of the posterior neural plate, with an anterior boundary similar
to that of hoxb1b. Restriction of hoxb1a expression to
presumptive r4 during somitogenesis is conserved in mouse
(Murphy and Hill, 1991), and
downregulation of hoxb1a might be necessary to allow normal
development of r5-r7. Although hoxb1a expression is activated
throughout the posterior hindbrain in a vhnf1-insensitive manner,
hoxb1a can be repressed by ectopic vhnf1 starting at tailbud
stage, the same stage at which hoxb1a transcripts are first
downregulated outside of r4. hoxb1a repression in future r5-r7 is
probably brought about at least in part by the function of vhnf1,
independent of Fgf and val functions. The separate roles of
vhnf1, as an activator in conjunction with Fgf signals and as a
repressor independent of Fgf signals, might reflect distinct molecular
interactions, either with cofactor proteins or with DNA. Two different forms
of Vhnf1 protein, which result from alternative splicing, have been
characterized as having different DNA binding affinities and transactivation
strengths (Ringeisen et al.,
1993
). It is possible that these isoforms provide the different
functional specificities in the caudal hindbrain.
Nonequivalence of vhnf1 and val functions
Although one of the central functions of vhnf1 in hindbrain
pattern formation is activation of val expression, loss of
vhnf1 has a more severe phenotype in the forming hindbrain than loss
of val function. Loss of val results in the production of a
narrowed `rX' domain in place of r5 and r6
(Moens et al., 1996). Like the
mis-specified r5 and r6 domain in vhnf1 mutant embryos, the
val mutant rX domain does not express hoxb3, and the
posterior boundary of hoxb1a and the anterior boundary of
hoxB4 expression are similarly indistinct
(Prince et al., 1998
).
However, the rX domain of val mutants is significantly narrower than
the combination of r5 and r6 domains, whereas vhnf1 mutants show no
apparent reduction of tissue. In addition, the reticulospinal neurons in
val mutants are correctly specified
(Moens et al., 1996
).
The genetic distinctions between vhnf1 and val are paralleled by differences in molecular capacities. The combination of val+Fgf is not sufficient to induce r5 identity outside the r5 domain, whereas the embryo is broadly sensitive to vhnf1+Fgf function. Conversely, Fgf+vhnf1 cannot induce ectopic krox20 in a val mutant background, suggesting that each transcription factor has unique and necessary functions in hindbrain pattern formation. Although overexpression of val in the vhnf1wi408 mutant background results in recovery of krox20 expression, this appears only in the r5 domain, with no ectopic krox20 expression detected. This suggests that only limited domains are competent to respond to val function, potentially based on the presence of a cofactor.
Potential conservation of vhnf1 function in hindbrain
pattern formation
Knockout of the murine vhnf1 gene results in early death as a
result of failure to form visceral endoderm
(Barbacci et al., 1999;
Coffinier et al., 1999
). As
yet, no studies of later loss-of-function of vhnf1 have been
published. However, vhnf1 is expressed in the mouse hindbrain in a
broad domain that lies close to the otic placode, a position that is similar
to the domain of expression in zebrafish
(Barbacci et al., 1999
;
Coffinier et al., 1999
). In
addition, vhnf1 expression is detected before the onset of
kreisler expression (Cordes and
Barsh, 1994
), suggesting that vhnf1 could have a
conserved role in regulation of kreisler expression in the developing
mouse hindbrain. Furthermore, expression of mouse hoxb1, the most
likely functional homolog of zebrafish hoxb1a, is downregulated in
the most posterior hindbrain at a time soon after initiation of vhnf1
transcription initiation (Murphy and Hill,
1991
). Finally, although fgf8 transcript expression is
restricted to r1 and the midbrain-hindbrain boundary (MHB) in mice,
fgf3 expression is observed in r4 at the same time that
kreisler expression is induced in presumptive r5+r6, and before the
upregulation of fgf3 in the r5+r6 domain
(Cordes and Barsh, 1994
;
Joyner et al., 2000
;
Mahmood et al., 1996
). Thus,
it is likely that the restriction of rhombomere-specific identities in the
caudal hindbrain of the mouse follows a molecular mechanism similar to the one
we have described for zebrafish.
Competence to respond to Fgf signals in the MHB and posterior
hindbrain
Fgf signals are reused throughout development and yet the cellular response
to the signal varies based on time and location. In the developing zebrafish
brain, fgf8 function is required not only for pattern formation in
the caudal hindbrain but also for formation of the MHB
(Reifers et al., 1998). In the
posterior hindbrain, Fgf signaling results in activation of val
expression, whereas, at the MHB, Fgf signaling results in activation of
gbx2, fkd3 and spry4
(Reim and Brand, 2002
). During
late gastrulation stages, the anterior hindbrain expresses fgf3 and
fgf8 in a domain that lies between the forming MHB and the posterior
hindbrain. Thus, it appears that the anterior hindbrain domain provides a
source of Fgf signals for both the MHB and posterior hindbrain. However, the
molecular and morphological results of this signaling are different.
Various features of Fgf signal transduction might provide distinct cellular
responses in the MHB and posterior hindbrain, including different Fgf receptor
interactions and negative feedback regulation of Fgf signal transduction. One
potential distinction in Fgf signaling outcome is the presence of
intracellular cofactors. We have shown here that the expression of
vhnf1 in or near cells receiving Fgf signals is sufficient to promote
activation of posterior hindbrain gene expression. Similarly, it has been
shown that the presence of pou2 in cells receiving Fgf signals is
required for activation of MHB target genes including gbx2, fkd3 and
spry4 (Reim and Brand,
2002). Thus, it is possible that one of the ways in which cells
generate a differential response to Fgf signals is through the presence of a
transcriptional cofactor such as pou2 or vhnf1, which
provides promoter selection specificity. Further work to characterize the
unique cellular responses to Fgf signals in the presence of pou2 or
vhnf1 will help determine the role of transcription factors as
mediators of signaling specificity.
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ACKNOWLEDGMENTS |
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Alexandre, D., Clarke, J. D., Oxtoby, E., Yan, Y. L., Jowett, T.
and Holder, N. (1996). Ectopic expression of
Hoxa-1 in the zebrafish alters the fate of the mandibular arch neural
crest and phenocopies a retinoic acid-induced phenotype.
Development 122,735
-746.
Barbacci, E., Reber, M., Ott, M. O., Breillat, C., Huetz, F. and
Cereghini, S. (1999). Variant hepatocyte nuclear
factor 1 is required for visceral endoderm specification.
Development 126,4795
-4805.
Bell, E., Wingate, R. J. and Lumsden, A.
(1999). Homeotic transformation of rhombomere identity after
localized Hoxb1 misexpression. Science
284,2168
-2171.
Coffinier, C., Thepot, D., Babinet, C., Yaniv, M. and Barra,
J. (1999). Essential role for the homeoprotein
vHNF1/HNF1ß in visceral endoderm differentiation.
Development 126,4785
-4794.
Cordes, S. P. and Barsh, G. S. (1994). The mouse segmentation gene kr encodes a novel basic domain-leucine zipper transcription factor. Cell 79,1025 -1034.[Medline]
Dupe, V. and Lumsden, A. (2001). Hindbrain patterning involves graded responses to retinoic acid signalling. Development 128,2199 -2208.[Medline]
Frohman, M. A., Martin, G. R., Cordes, S. P., Halamek, L. P. and
Barsh, G. S. (1993). Altered rhombomere-specific gene
expression and hyoid bone differentiation in the mouse segmentation mutant,
kreisler (kr). Development
117,925
-936.
Gavalas, A. and Krumlauf, R. (2000). Retinoid signalling and hindbrain patterning. Curr. Opin. Genet. Dev. 10,380 -386.[CrossRef][Medline]
Gavalas, A., Studer, M., Lumsden, A., Rijli, F. M., Krumlauf, R.
and Chambon, P. (1998). Hoxa1 and Hoxb1 synergize in
patterning the hindbrain, cranial nerves and second pharyngeal arch.
Development 125,1123
-1136.
Giudicelli, F., Gilardi-Hebenstreit, P., Mechta-Grigoriou, F., Poquet, C. and Charnay, P. (2003). Novel activities of Mafb underlie its dual role in hindbrain segmentation and regional specification. Dev. Biol. 253,150 -162.[CrossRef][Medline]
Griffin, K. J., Amacher, S. L., Kimmel, C. B. and Kimelman,
D. (1998). Molecular identification of spadetail:
regulation of zebrafish trunk and tail mesoderm formation by T-box genes.
Development 125,3379
-3388.
Joyner, A. L., Liu, A. and Millet, S. (2000). Otx2, Gbx2 and Fgf8 interact to position and maintain a mid-hindbrain organizer. Curr. Opin. Cell Biol. 12,736 -741.[CrossRef][Medline]
Kolm, P. J. and Sive, H. L. (1995). Regulation of the Xenopus labial homeodomain genes, HoxA1 and HoxD1: activation by retinoids and peptide growth factors. Dev. Biol. 167,34 -49.[CrossRef][Medline]
Koshida, S., Shinya, M., Mizuno, T., Kuroiwa, A. and Takeda,
H. (1998). Initial anteroposterior pattern of the zebrafish
central nervous system is determined by differential competence of the
epiblast. Development
125,1957
-1966.
Kudoh, T., Wilson, S. W. and Dawid, I. B. (2002). Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm. Development 129,4335 -4346.[Medline]
Lumsden, A. and Krumlauf, R. (1996). Patterning
the vertebrate neuraxis. Science
274,1109
-1115.
Mahmood, R., Mason, I. J. and Morriss-Kay, G. M. (1996). Expression of Fgf-3 in relation to hindbrain segmentation, otic pit position and pharyngeal arch morphology in normal and retinoic acid-exposed mouse embryos. Anat. Embryol. 194, 13-22.[Medline]
Manley, N. R. and Capecchi, M. R. (1997). Hox group 3 paralogous genes act synergistically in the formation of somitic and neural crest-derived structures. Dev. Biol. 192,274 -288.[CrossRef][Medline]
Manzanares, M., Trainor, P. A., Nonchev, S., Ariza-McNaughton, L., Brodie, J., Gould, A., Marshall, H., Morrison, A., Kwan, C. T., Sham, M. H. et al. (1999). The role of kreisler in segmentation during hindbrain development. Dev. Biol. 211,220 -237.[CrossRef][Medline]
Marin, F. and Charnay, P. (2000). Hindbrain
patterning: FGFs regulate Krox20 and mafB/kr
expression in the otic/preotic region. Development
127,4925
-4935.
Maves, L., Jackman, W. and Kimmel, C. B. (2002). FGF3 and FGF8 mediate a rhombomere 4 signaling activity in the zebrafish hindbrain. Development 129,3825 -3837.[Medline]
McClintock, J. M., Kheirbek, M. A. and Prince, V. E. (2002). Knockdown of duplicated zebrafish hoxb1 genes reveals distinct roles in hindbrain patterning and a novel mechanism of duplicate gene retention. Development 129,2339 -2354.[Medline]
Moens, C. B. and Prince, V. E. (2002). Constructing the hindbrain: insights from the zebrafish. Dev. Dyn. 224,1 -17.[CrossRef][Medline]
Moens, C. B., Yan, Y. L., Appel, B., Force, A. G. and Kimmel, C.
B. (1996). valentino: a zebrafish gene required for
normal hindbrain segmentation. Development
122,3981
-3990.
Moens, C. B., Cordes, S. P., Giorgianni, M. W., Barsh, G. S. and
Kimmel, C. B. (1998). Equivalence in the genetic
control of hindbrain segmentation in fish and mouse.
Development 125,381
-391.
Murphy, P. and Hill, R. E. (1991). Expression of the mouse labial-like homeobox-containing genes, Hox 2.9 and Hox 1.6, during segmentation of the hindbrain. Development 111,61 -74.[Abstract]
Oxtoby, E. and Jowett, T. (1993). Cloning of the zebrafish krox-20 gene (krx- 20) and its expression during hindbrain development. Nucleic Acids Res. 21,1087 -1095.[Abstract]
Power, S. C. and Cereghini, S. (1996). Positive regulation of the vHNF1 promoter by the orphan receptors COUP-TF1/Ear3 and COUP-TFII/Arp1. Mol. Cell. Biol. 16,778 -791.[Abstract]
Prince, V. E., Moens, C. B., Kimmel, C. B. and Ho, R. K.
(1998). Zebrafish Hox genes: expression in the hindbrain region
of wild-type and mutants of the segmentation gene, valentino.
Development 125,393
-406.
Raible, F. and Brand, M. (2001). Tight transcriptional control of the ETS domain factors Erm and Pea3 by Fgf signaling during early zebrafish development. Mech. Dev. 107,105 -117.[CrossRef][Medline]
Reifers, F., Bohli, H., Walsh, E. C., Crossley, P. H., Stainier,
D. Y. and Brand, M. (1998). Fgf8 is mutated in
zebrafish acerebellar (ace) mutants and is required for
maintenance of midbrain-hindbrain boundary development and somitogenesis.
Development 125,2381
-2395.
Reim, G. and Brand, M. (2002). Spiel-ohne-grenzen/pou2 mediates regional competence to respond to Fgf8 during zebrafish early neural development. Development 129,917 -933.[Medline]
Ringeisen, F., Rey-Campos, J. and Yaniv, M.
(1993). The transactivation potential of variant hepatocyte
nuclear factor 1 is modified by alternative splicing. J. Biol.
Chem. 268,25706
-25711.
Rodaway, A., Takeda, H., Koshida, S., Broadbent, J., Price, B.,
Smith, J. C., Patient, R. and Holder, N. (1999).
Induction of the mesendoderm in the zebrafish germ ring by yolk cell-derived
TGF-beta family signals and discrimination of mesoderm and endoderm by FGF.
Development 126,3067
-3078.
Rossel, M. and Capecchi, M. R. (1999). Mice
mutant for both Hoxa1 and Hoxb1 show extensive remodeling of
the hindbrain and defects in craniofacial development.
Development 126,5027
-5040.
Sagerstrom, C. G., Grinbalt, Y. and Sive, H.
(1996). Anteroposterior patterning in the zebrafish, Danio
rerio: an explant assay reveals inductive and suppressive cell
interactions. Development
122,1873
-1883.
Sagerstrom, C. G., Kao, B. A., Lane, M. E. and Sive, H. (2001). Isolation and characterization of posteriorly restricted genes in the zebrafish gastrula. Dev. Dyn. 220,402 -408.[CrossRef][Medline]
Salzberg, A., Elias, S., Nachaliel, N., Bonstein, L., Henig, C. and Frank, D. (1999). A Meis family protein caudalizes neural cell fates in Xenopus. Mech. Dev. 80, 3-13.[CrossRef][Medline]
Schulte-Merker, S., Ho, R. K., Herrmann, B. G. and
Nusslein-Volhard, C. (1992). The protein product of the
zebrafish homologue of the mouse T gene is expressed in nuclei of the germ
ring and the notochord of the early embryo.
Development 116,1021
-1032.
Seitanidou, T., Schneider-Maunoury, S., Desmarquet, C., Wilkinson, D. G. and Charnay, P. (1997). Krox-20 is a key regulator of rhombomere-specific gene expression in the developing hindbrain. Mech. Dev. 65,31 -42.[CrossRef][Medline]
Studer, M., Gavalas, A., Marshall, H., Ariza-McNaughton, L.,
Rijli, F. M., Chambon, P. and Krumlauf, R. (1998).
Genetic interactions between Hoxa1 and Hoxb1 reveal new
roles in regulation of early hindbrain patterning.
Development 125,1025
-1036.
Sun, Z. and Hopkins, N. (2001). vhnf1,
the MODY5 and familial GCKD-associated gene, regulates regional specification
of the zebrafish gut, pronephros, and hindbrain. Genes
Dev. 15,3217
-3229.
Trainor, P. A. and Krumlauf, R. (2001). Hox genes, neural crest cells and branchial arch patterning. Curr. Opin. Cell Biol. 13,698 -705.[CrossRef][Medline]
Vlachakis, N., Choe, S. K. and Sagerstrom, C. G.
(2001). Meis3 synergizes with Pbx4 and Hoxb1b in promoting
hindbrain fates in the zebrafish. Development
128,1299
-1312.
Walshe, J., Maroon, H., McGonnell, I. M., Dickson, C. and Mason, I. (2002). Establishment of hindbrain segmental identity requires signaling by FGF3 and FGF8. Curr. Biol. 12,1117 -1123.[CrossRef][Medline]
Waskiewicz, A. J., Rikhof, H. A., Hernandez, R. E. and Moens, C.
B. (2001). Zebrafish Meis functions to stabilize Pbx proteins
and regulate hindbrain patterning. Development
128,4139
-4151.
Waskiewicz, A. J., Rikhof, H. A. and Moens, C. B. (2002). Eliminating zebrafish Pbx proteins reveals a hindbrain ground state. Dev. Cell 3, 723-733.[Medline]
Weinberg, E. S., Allende, M. L., Kelly, C. S., Abdelhamid, A.,
Murakami, T., Andermann, P., Doerre, O. G., Grunwald, D. J. and
Riggleman, B. (1996). Developmental regulation of zebrafish
MyoD in wild-type, no tail and spadetail embryos.
Development 122,271
-280.
Westerfield, M. (1995). The Zebrafish Book. Eugene, Oregon: University of Oregon Press.
Woo, K. and Fraser, S. E. (1997). Specification
of the zebrafish nervous system by nonaxial signals.
Science 277,254
-257.
Woo, K. and Fraser, S. E. (1998). Specification of the hindbrain fate in the zebrafish. Dev. Biol. 197,283 -296.[CrossRef][Medline]