1 OncoDevelopmental Biology Program, Burnham Institute, 10901 North Torrey Pines
Road, La Jolla, CA 92037, USA
2 Unité d'Expression Génétique et Maladie URA 1644,
Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France
3 Unité de Biologie du Développement URA 2578, Institut Pasteur,
25 rue du Docteur Roux, 75724 Paris Cedex 15, France
* Author for correspondence (e-mail: duester{at}burnham.org)
Accepted 1 April 2005
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SUMMARY |
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Key words: Retinoic acid, Hindbrain, Segmentation, Raldh2 (Aldh1a2), Cyp26, Hoxb1, Hnf1 (Tcf2), Mouse
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Introduction |
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The transcriptional regulation of Hox genes has been examined extensively
as previously reviewed (Lufkin,
1996). It has been discovered that the mouse Hoxb1 gene
is regulated by a retinoic acid response element (RARE) located 3' to
the promoter that is required for early widespread induction in the posterior
hindbrain up to the presumptive r3/4 boundary
(Marshall et al., 1994
). This
is consistent with the more recent demonstration that Hoxb1
expression anterior to the node requires cell to cell signaling and does not
rely on proliferative expansion of Hoxb1-expressing cells at the
level of the node (Forlani et al.,
2003
). Interestingly, another RARE located 5' to the
promoter has been demonstrated to be required for repression of Hoxb1
in r3 and r5 to provide restricted expression in r4
(Studer et al., 1994
). In
addition, an autoregulatory element has been found in the Hoxb1
promoter that is important for maintenance of r4 expression
(Pöpperl et al., 1995
).
However, RA activity has not previously been detected anterior to r5 (see
below) and other factors have been found to regulate Hoxb1 r4
restriction. In zebrafish, repression of Hoxb1 in the posterior
hindbrain up to r5 has been demonstrated to depend upon the homeodomain
protein encoded by vHnf1 (variant of Hepatocyte nuclear factor 1;
Tcf2 Mouse Genome Informatics), which is expressed in the
posterior hindbrain up to the r4/r5 boundary
(Wiellette and Sive, 2003
).
Recent studies also indicate that RA is required for expression of zebrafish
vHnf1 in the posterior hindbrain
(Hernandez et al., 2004
). In
addition, zebrafish iro7 encodes an iroquois homeodomain protein
(homologous to mouse Irx3) expressed in the anterior hindbrain down
to r4, and mutual repression of iro7 and vHnf1 positions the
r4/r5 boundary (Lecaudey et al.,
2004
). Thus, it is unclear if mouse Hoxb1 induction and
r4 restriction involves direct effects of RA signaling on the Hoxb1
promoter as RA has not been detected anterior to r5, plus it is unknown if RA
may have indirect effects on mouse Hoxb1 r4 restriction through
regulation of vHnf1 or Irx3 to set the r4/r5 boundary.
In the mouse, RA is first generated at embryonic day 7.5 (E7.5) just prior
to the onset of hindbrain development
(Rossant et al., 1991;
Ang et al., 1996
). RA synthesis
for mouse development between E7.5-E8.25 is controlled by retinaldehyde
dehydrogenase 2, encoded by Raldh2 (Aldh1a2 Mouse
Genome Informatics), expressed in the trunk paraxial mesoderm destined to
become somites (Niederreither et al.,
1999
; Mic et al.,
2002
). The timing and location of the initial expression of
Raldh2 coincides with the onset of posterior neural development, and
RA as well as Raldh2 are indeed required for posterior hindbrain
development and Hoxb1 expression in amniote embryos
(Maden et al., 1996
;
Dickman et al., 1997
;
White et al., 1998
;
Niederreither et al., 2000
;
Dupé and Lumsden, 2001
)
as well as Xenopus (Blumberg et
al., 1997
; Kolm et al.,
1997
; Van der Wees et al.,
1998
; Chen et al.,
2001
) and zebrafish embryos
(Begemann et al., 2001
;
Grandel et al., 2002
;
Kudoh et al., 2002
). This
suggests that RA generated in the trunk paraxial mesoderm by Raldh2
travels anteriorly into the hindbrain. It has been further suggested that a
gradient of RA may exist across the hindbrain with the high point located
posteriorly where the paraxial mesoderm lies adjacent to the posterior
hindbrain. Examination of RA activity in mouse embryos carrying an RA-reporter
transgene (RARE-lacZ) has revealed that RA activity is limited to the
posterior portion of the embryo at the late primitive streak stage (E7.5)
(Rossant et al., 1991
), but
the exact location of its anterior limit was not determined. At E8.25-E9.25,
the RARE-lacZ RA signal has been shown to be present in the posterior
hindbrain up to r5 (Sakai et al.,
2001
; Mic et al.,
2002
), but there has been no clear indication that RA activity is
ever present further anterior in mouse embryos. This issue has not been
resolved in other vertebrate embryos because of the lack of an appropriate RA
reporter. As RA activity has not been convincingly demonstrated in r3 and r4,
it remains unclear whether the Hoxb1 RARE DNA control elements
described above require RA to function in those segments. A failure to observe
RA activity in r3 and r4 may be due to expression of RA-degrading P450s in the
hindbrain. Indeed, three RA-degrading P450s encoded by Cyp26a1
(Fujii et al., 1997
;
Hollemann et al., 1998
;
Swindell et al., 1999
;
Kudoh et al., 2002
),
Cyp26b1 (MacLean et al.,
2001
; Reijntjes et al.,
2003
) and Cyp26c1
(Tahayato et al., 2003
;
Reijntjes et al., 2004
) are
expressed in dynamic patterns during hindbrain development in several
vertebrate embryos.
We now provide further insight into the mechanism of RA action during
establishment of r3/r4/r5 gene expression boundaries through analysis of
RARE-lacZ, Raldh2/ and
vHnf1/ mouse embryos. We demonstrate the
existence of dynamic shifting boundaries of hindbrain RA activity during
Hoxb1 induction/repression that correspond to the Cyp26a1
and Cyp26c1 expression patterns. We show that RA is transiently
present throughout the posterior hindbrain up to the r2/r3 boundary (abutting
the anterior Cyp26a1 expression domain) and that this RA is needed to
induce Hoxb1 expression throughout the posterior hindbrain up to the
presumptive r3/r4 boundary and to induce vHnf1 (a repressor of
Hoxb1) up to the presumptive r4/r5 boundary. However, subsequent to
induction of Hoxb1 and vHnf1, the boundary of RA activity is
quickly shifted to r4/r5, coincident with initiation of Cyp26c1
expression in r4, and this coincides with strict limitation of Irx3
and vHnf1 expression to opposite sides of the r4/r5 boundary, plus
restriction of Hoxb1 expression to r4. Studies on
Raldh2/ and
vHnf1/ embryos indicate that RA is required
for induction of Hoxb1 and vHnf1, and that vHnf1 is
required to repress Hoxb1 posterior to r4. Analysis of RA-treated
embryos supports a functional role for Cyp26c1 in RA degradation. Our
findings thus provide evidence that RA activity exists in the appropriate
location to directly induce Hoxb1 throughout the posterior hindbrain
and to directly repress Hoxb1 in r3 and r5 through previously
described 3' and 5' RAREs
(Marshall et al., 1994;
Studer et al., 1994
). We also
demonstrate that RA-mediated repression of Hoxb1 posterior to r4 also
functions indirectly through RA induction of its repressor vHnf1.
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Materials and methods |
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In order to examine vHnf1/ embryos during
hindbrain development, we used a conditional knockout of vHnf1
described previously (Coffinier et al.,
2002). We also used the Mox2-Cre (MORE) transgene, which
expresses Cre throughout the epiblast following implantation but not in
extra-embryonic tissues (Tallquist and
Soriano, 2000
). Conditional
vHnf1/ embryos were generated from matings
between mice homozygous for a conditional vHnf1 allele flanked by
loxP sites with mice that were heterozygous for the null allele of
vHnf1 (Coffinier et al.,
1999
) and also carried a Mox2-Cre allele.
Embryo genotyping and staging
Embryos from timed matings were genotyped by PCR analysis of yolk sac DNA.
Embryos were staged according to morphology as previously described
(Downs and Davies, 1993;
Forlani et al., 2003
) and were
assigned the following embryonic day numbers with noon on the day of vaginal
plug detection being considered embryonic day 0.5 (E0.5): early headfold
(E7.4), headfold (E7.5-E7.75), late headfold (E7.8-7.9), one to three somites
(E8.0), four to six somites (E8.25), seven to ten somites (E8.5) and 11-14
somites (E8.75).
Rescue of mutant embryos with a physiological dose of RA
Rescue of Raldh2/ embryos by maternal
dietary RA supplementation was performed as previously described with an RA
dose that has previously been demonstrated to be in the normal physiological
range (Mic et al., 2003).
Briefly, all-trans-RA (Sigma) was dissolved in corn oil and mixed
with powdered mouse chow to provide a final concentration of 0.1 mg/g for
treatment from E6.75-E8.25 or from E6.75 to the point of analysis for embryos
analyzed prior to E8.25. Such food was prepared fresh twice each day (morning
and evening) and provided ad libitum. For mice analyzed after E8.25, mice were
returned to standard mouse chow at E8.25 until the point of analysis.
Treatment of wild-type embryos with excess RA
Pregnant wild-type mice were treated with exogenous RA similar to a
previous description (Conlon and Rossant,
1992). Following timed matings, female mice were orally
administered all-trans-RA (Sigma) at a dose of 20 mg/kg body weight
dissolved in 0.2 ml corn oil at 12 pm on day 7 (E7.5). Embryos were analyzed
18 hours after dosing (E8.25).
Whole-mount in situ hybridization
Embryonic mRNAs were detected by whole-mount in situ hybridization using
the alkaline phosphatase substrate NBT-BCIP as described
(Wilkinson, 1992). Antisense
RNA probes were generated from mouse cDNAs encoding Raldh2
(Haselbeck et al., 1999
),
Hoxb1 (Hunt et al.,
1991
), vHnf1
(Coffinier et al., 1999
),
Irx3 (Cohen et al.,
2000
), Cyp26a1 and Cyp26c1
(Tahayato et al., 2003
),
Krox20 (Wilkinson,
1993
), and Epha2
(Becker et al., 1994
).
Detection of retinoic acid activity
Detection of RA activity was performed in embryos carrying the
RARE-lacZ RA-reporter transgene, which places lacZ (encoding
ß-galactosidase) under the transcriptional control of a retinoic acid
response element (RARE) (Rossant et al.,
1991). ß-Galactosidase staining was performed for 1 hour with
the substrate Salmon-gal (6-chloro-3-indolyl-ß-D-galactopyranoside)
(Labscientific) to produce a red reaction product; in some cases, staining was
performed with the substrate X-gal
(5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) to produce a
blue-green reaction product. Double staining to examine both RA activity
(RARE-lacZ) and mRNA localization was performed by first staining 1
hour for ß-galactosidase using Salmon-gal, followed by processing for
whole-mount in situ hybridization as described
(Tajbakhsh and Houzelstein,
1995
).
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Results |
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In zebrafish embryos, vHnf1 has been found to be expressed in the
posterior hindbrain up to the r4/r5 boundary
(Wiellette and Sive, 2003;
Hernandez et al., 2004
). In
mouse embryos, vHnf1 expression has been reported in the posterior
hindbrain at E8.0-E8.5 (Coffinier et al.,
1999
; Barbacci et al.,
1999
), but its anterior boundary has not been defined. E8.0 mouse
embryos double stained for vHnf1 and Krox20 (which is
limited to r3 at E8.0) exhibited a gap between the two domains, indicating
that vHnf1 is not expressed in r4
(Fig. 1F). A similar gap was
observed at E8.25 when Krox20 is expressed strongly in r3 and weakly
in r5, indicating that vHnf1 expression overlaps the r5 domain of
Krox20 (Fig. 1G; also
see Fig. 2M for Krox20
r5 expression). Double-staining for Epha2 (which is limited to r4)
and vHnf1 revealed that their expression domains meet without a gap
(Fig. 1H). These results
indicate that vHnf1 is expressed in the posterior hindbrain up to the
r4/r5 boundary.
The zebrafish iro7 gene is expressed in the anterior hindbrain
down to the r4/r5 boundary (Lecaudey et
al., 2004). The mouse homolog, Irx3, is also expressed in
the anterior neural plate (Bosse et al.,
1997
), but its anterior boundary is not defined. At E8.25,
Irx3 was expressed in the anterior hindbrain down to approximately
the r4/r5 boundary (Fig. 1I)
when compared with the previously established r5 expression border of
vHnf1 at E8.25 (Fig.
1G). Double-staining for Irx3 and vHnf1
expression at E8.0 provided further evidence that the posterior border of
Irx3 expression is in r4 as it lies directly adjacent to the
vHnf1 r5 domain (Fig.
1J).
Shifting boundaries of RA activity along the hindbrain
RA synthesis during early mouse hindbrain development is controlled by
Raldh2 expressed in the paraxial mesoderm
(Niederreither et al., 2000),
and RA degradation is controlled at least in part by Cyp26a1
expressed in the anterior neural plate
(Sakai et al., 2001
;
Abu-Abed et al., 2001
). A
similar situation exists in Xenopus, chick and zebrafish embryos
(Hollemann et al., 1998
;
Swindell et al., 1999
;
Kudoh et al., 2002
). This has
led to the hypothesis that RA synthesized in the paraxial mesoderm may diffuse
anteriorly across the hindbrain until it meets the Cyp26a1 expression
domain, perhaps establishing a posterior-high gradient of RA activity across
the hindbrain. Such RA is presumably needed for induction of various genes
including Hoxb1 whose expression in r4 requires two retinoic acid
response elements (RAREs) located 3' and 5' of the promoter
(Marshall et al., 1994
;
Studer et al., 1994
). Among
the various vertebrate embryo models of hindbrain development, the mouse is
unique in that an RA-reporter transgene (RARE-lacZ) has been
constructed that allows RA activity to be detected during the early stages of
hindbrain development (Rossant et al.,
1991
). RARE-lacZ expression is completely eliminated in
the hindbrain of Raldh2/ embryos and can be
induced in all embryonic cells of wild-type embryos following treatment with
excess RA, thus demonstrating that this transgene is indeed a faithful
reporter for endogenous RA (Niederreither
et al., 1999
; Mic et al.,
2002
). Previous studies using RARE-lacZ embryos have not
defined the anterior boundary of RA activity during the headfold stages,
although it is clear that RA is present in the hindbrain up to the r5 boundary
at E8.25-E9.25 (Sakai et al.,
2001
; Mic et al.,
2002
). Here, we have examined wild-type headfold stage embryos to
determine if RA exists anterior to r5.
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RA induction of Cyp26c1 in r4
Although the expression domain of Cyp26a1 is positioned properly
to account for the formation of an r2/r3 boundary of RA activity, it cannot
account for the subsequent shift to r4/r5. Previous studies have shown that a
related enzyme encoded by Cyp26c1 is expressed in r2 and r4 as well
as the adjacent head mesoderm in E8.0-E8.5 mouse embryos
(Tahayato et al., 2003). A
third related mouse enzyme encoded by Cyp26b1 is expressed in r3 and
r5 at approximately E8.25, but this is clearly after Cyp26c1
expression is already apparent in r4
(MacLean et al., 2001
) (I.O.S.
and G.D., unpublished).
|
RA is required for early expansion of Hoxb1 expression to r3/r4 border
RA generated by Raldh2 has previously been shown to be required
for r4 expression of Hoxb1 in E8.25-E8.5 mouse embryos
(Niederreither et al., 2000).
We examined the effect of a loss of RA synthesis on the early anterior
expansion of Hoxb1 expression. The normal expansion of Hoxb1
expression from the node to the r3/r4 boundary is shown in wild-type embryos
examined from E7.5-E8.0 (Fig.
4A-C). By E8.0, Hoxb1 is continuously expressed from the
node to r3/r4 and has not yet been restricted to r4
(Fig. 4C).
Raldh2/ embryos at E8.0 completely lacked
the anterior expansion of Hoxb1 to r3/r4, thus resembling wild-type
E7.5 embryos in which Hoxb1 expression was limited anteriorly to a
region near the node (n=3; Fig.
4A,C). These findings demonstrate that RA generated by
Raldh2 is required for the initial expansion of Hoxb1
expression throughout the hindbrain up to r3/r4.
|
Raldh2/ embryos at E8.25-E8.5 lacked the r4 domain of Hoxb1 expression, but exhibited residual Hoxb1 expression at the posterior hindbrain/spinal cord junction that would normally be eliminated by this stage (n=3; Fig. 4I-L). Raldh2/ embryos double stained for Cyp26c1 and Hoxb1 demonstrated a complete loss of expression of both genes in r4, but retention of Hoxb1 expression at the posterior hindbrain/spinal cord junction lying just posterior to the remaining head mesodermal domain of Cyp26c1 (Fig. 4K-L). This residual posterior domain of Hoxb1 expression may be the remnants of that observed near the node in E8.0 Raldh2/ embryos (Fig. 4C). These findings suggest that mouse vHnf1 may function in repression of Hoxb1, as the loss of vHnf1 observed in Raldh2/ embryos may result in a failure to repress this RA-independent posterior domain of Hoxb1 expression.
Raldh2/ embryos examined at E7.9-E8.25 exhibited a posterior expansion of Irx3 expression in the hindbrain (n=6; Fig. 4M-O). Whereas Irx3 expression in the anterior hindbrain normally displayed an r4 posterior boundary, a loss of RA synthesis resulted in an expansion posteriorly throughout the hindbrain to the spinal cord junction. This abnormal posterior domain of Irx3 expression lies at approximately the same location where Hoxb1 expression is abnormally retained in the mutant posterior hindbrain (compare Fig. 4J with Fig. 4O). These results suggest that the effect of RA on mouse Irx3 expression may be due to the requirement of RA for expression of vHnf1, which may function (as it does in zebrafish) as a repressor of both Irx3 and Hoxb1 in the posterior hindbrain up to the r4/r5 boundary.
Requirement of vHnf1 for r4/r5 gene expression boundary
We examined the effect of a loss of vHnf1 function on expression
of Hoxb1 in the mouse hindbrain. Disruption of mouse vHnf1
has previously been shown to result in an extra-embryonic defect of visceral
endoderm formation, leading to embryonic lethality prior to hindbrain
formation (Coffinier et al.,
1999; Barbacci et al.,
1999
). A conditional vHnf1 mutant mouse has allowed the
function of this gene to be examined at later stages
(Coffinier et al., 2002
).
Here, conditional vHnf1/ embryos were
generated from matings with mice containing the Mox2-Cre (MORE)
transgene, which stimulates Cre/lox-mediated deletion throughout the epiblast
following implantation, but does not affect extra-embryonic tissues
(Tallquist and Soriano, 2000
).
Hoxb1 expression in E8.5 conditional
vHnf1/ embryos was observed posterior to the
normal r4/r5 boundary, indicating a failure to restrict Hoxb1
expression to r4 (n=3; Fig.
5A-B). These findings indicate that the function of vHnf1
as a Hoxb1 repressor is conserved in mouse and zebrafish.
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|
Excess RA induces anterior shifts in Cyp26c1 domain and Hoxb1/vHnf1 boundary
Our results suggest that Cyp26c1 may provide an RA degradation
function in r4 to limit the anterior border of RA activity, and that this
border may be crucial for establishing a border between Hoxb1 and
vHnf1 at the r4/r5 boundary. Genetic studies of the related genes
Cyp26a1 and Cyp26b1 have revealed that both have a function
in RA degradation (Sakai et al.,
2001; Abu-Abed et al.,
2001
; Yashiro et al.,
2004
), but genetic studies for Cyp26c1 have not been
reported. In order to test this potential function for Cyp26c1, we
treated RARE-lacZ embryos with a teratogenic dose of RA to disrupt
the normal boundary of RA activity. Previous studies have shown that a 20
mg/kg dose of RA administered at E7.5 leads to induction of RARE-lacZ
expression throughout the anterior region of the embryo when examined 6 hours
after treatment (Rossant et al.,
1991
), thus eliminating the normal RA boundary in the hindbrain.
When we administered a 20 mg/kg dose of RA at E7.5, we found that some embryos
examined at E8.25 (18 hours after treatment) still exhibited a shift in
RARE-lacZ expression completely to the anterior tip
(n=9/15), but that some embryos exhibited clearing of
RARE-lacZ expression at the anterior-most region of the embryo
(n=6/15) and that all embryos exhibited reduced RARE-lacZ
expression posteriorly in the tailbud (Fig.
7A). Cyp26a1 expression following this treatment was
observed in the tailbud where RARE-lacZ expression was reduced,
consistent with its known function in RA degradation (n=3;
Fig. 7B); expression was
similar to that in untreated embryos, which normally downregulate
Cyp26a1 anteriorly and upregulate expression in the tailbud by E8.25
(MacLean et al., 2001
).
However, this RA treatment resulted in a shift in Cyp26c1 expression
to the anterior-most region of the embryo (n=7;
Fig. 7C; compare with untreated
control in Fig. 3F). A closer
comparison demonstrates that this new Cyp26c1 expression domain lies
in approximately where RARE-lacZ expression first begins to clear
anteriorly (Fig. 7D-E). This
provides evidence that Cyp26c1 is functioning to degrade RA in this
anteriorly shifted domain.
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Discussion |
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Previous studies on chick embryos treated with RA receptor antagonists
provided evidence that hindbrain patterning requires graded responses to RA,
with a higher RA concentration being required posteriorly
(Dupé and Lumsden,
2001). Our findings with mouse embryos demonstrate that the length
of RA exposure is also important for regulating hindbrain gene expression as
RA activity was observed for only 6-8 hours in r3-r4 (E7.6-E7.9), but for at
least 20 hours in r5-r8 (E7.6-E8.5). The studies we present also make it clear
that a stable RA gradient is not established across the hindbrain, but that
the initial gradient of RA entering the posterior hindbrain is converted by
RA-degrading enzymes into RA boundaries that shift over time such that
anterior tissues receive a short pulse of RA and posterior tissues receive a
long pulse of RA.
|
It is unclear how RA-liganded RA receptors bound to the 5' RARE of
Hoxb1 result in r3/r5 repression whereas RA-liganded RA receptors
bound to the 3' RARE of Hoxb1 stimulate widespread induction in
the posterior hindbrain up to r4. It is possible that additional
transcriptional regulatory proteins expressed in r3 and/or r5 bind near the
5' RARE and interact with an RA-liganded RA receptor in such a fashion
that results in Hoxb1 repression in r3 and r5 rather than induction.
In addition, as a loss of Cyp26a1 function in mouse embryos results
in ectopic expression of Hoxb1 in r3 (but not r5) and presumably
higher RA activity in r3 (Sakai et al.,
2001; Abu-Abed et al.,
2001
), this suggests that the 5' RARE is insufficient to
repress Hoxb1 in r3. Perhaps the relative activities of the 3'
and 5' RAREs are determined by levels of RA such that a higher than
normal level of RA in r3 disrupts 5' RARE repression and/or allows
3' RARE induction of Hoxb1.
RA signaling is required for neural expression of vHnf1
Our findings shed more light on the mechanism whereby vHnf1 is
induced in the posterior hindbrain. We find that RA activity is required for
induction of mouse vHnf1 as previously reported for zebrafish
vHnf1 (Hernandez et al.,
2004). However, other previous studies have suggested that
Hoxb1 may be sufficient to induce vHnf1 in zebrafish
(Choe and Sagerström,
2004
). Our studies suggest that Hoxb1 is not sufficient
for vHnf1 induction as we find persistent expression of
Hoxb1 in the posterior hindbrain and anterior spinal cord of
Raldh2/ embryos, but a complete lack of
vHnf1 expression in this domain of Hoxb1 expression. Thus,
we suggest that RA signaling is needed to activate vHnf1. A direct
effect of RA is plausible as a DR1 retinoid response element has been
identified in the promoter region of mouse vHnf1
(Power and Cereghini, 1996
).
As the vHnf1 DR1 response element was found to bind retinoid
receptors less efficiently than a DR5 retinoid response element (such as those
present near the RARß and Hoxb1 genes), it was suggested that
vHnf1 may be less responsive to RA than genes with DR5 retinoid
response elements (Power and Cereghini,
1996
). We suggest that this lower responsiveness of vHnf1
to RA may be crucial to the mechanism whereby its expression domain is limited
to a more posterior boundary than that of Hoxb1. The r5 anterior
limit of vHnf1 expression is in fact closer to the paraxial
mesodermal source of RA than the r4 anterior limit of Hoxb1, and we
have demonstrated that RA activity is transient in r4 but more long lived in
r5.
Hoxb1 expression in the absence of RA activity
Studies on quail embryos reported that vitamin A deficiency (VAD) results
in a complete absence of r4-r8, with the hindbrain consisting of an enlarged
r1-r3 (devoid of Hoxb1 expression) joined to the anterior spinal
cord, which retained expression of Hoxb1
(Maden et al., 1996). A
similar phenotype was also observed in mouse
Raldh2/ embryos at E8.25-E8.5, which lacked
the characteristic r4 stripe of Hoxb1 expression, but retained a
diffuse domain of Hoxb1 expression in the posterior-most region of
the hindbrain next to the spinal cord junction
(Niederreither et al., 2000
).
However, results from chick embryos treated with retinoid receptor antagonists
suggested that RA deficiency leads to elimination of r5-r8 but with an
enlarged r4 remaining because of residual expression of Hoxb1 and
other r4 markers in the posterior-most region of the hindbrain adjacent to the
spinal cord (Dupé and Lumsden,
2001
). Results from VAD rat embryos also suggested that r4 is not
totally eliminated (Baybutt et al.,
2000
). The residual Hoxb1 expression observed in the
posterior hindbrain of mouse Raldh2/ embryos
was originally not interpreted as an indication of r4 character, possibly
owing to the observation of an extension of the Krox20 r3 expression
domain all the way to the spinal cord junction
(Niederreither et al., 2000
).
We note that the r4 expression domain of Cyp26c1 is completely
missing in the posterior hindbrain of
Raldh2/ embryos (but an expanded r2
expression domain was observed), providing further evidence that at least some
aspects of r4 character have been lost in the absence of RA activity. In
addition, residual expression of Hoxb1 in the posterior hindbrain of
Raldh2/ embryos may be due to the loss of
vHnf1 expression, which normally functions to repress Hoxb1
in that location. Rather than being an indicator of r4 character, this
residual Hoxb1 expression may be the remnants of that which normally
occurs independent of RA in posterior neuroectoderm up to the level of the
node prior to anterior expansion of Hoxb1 expression into the
hindbrain (Forlani et al.,
2003
).
Conserved function for RA boundaries
The model of mouse hindbrain RA activity proposed here is likely to be
conserved in other vertebrate embryos. Raldh2 expression occurs in
the trunk paraxial mesoderm of all vertebrate embryos analyzed and
Cyp26 homologs expressed in the hindbrain exist as well (see
Introduction). Studies on zebrafish embryos have demonstrated that
vHnf1 functions as a repressor of Hoxb1 in r5
(Wiellette and Sive, 2003) and
it was recently reported that RA is required for vHnf1 expression in
the zebrafish hindbrain (Hernandez et al.,
2004
). In addition, zebrafish Irx3 (iro7) and
vHnf1 function as mutual repressors needed to establish an r4/r5
expression boundary (Lecaudey et al.,
2004
). Thus, shifting boundaries of RA activity that regulate the
spatiotemporal expression patterns of Hoxb1, vHnf1 and
Irx3 may be a general feature of vertebrate hindbrain development.
However, there have been no reports of methods to localize RA activity in the
hindbrain of other vertebrates to directly test this. Thus, our studies
highlight the importance of using mouse embryos carrying the
RARE-lacZ transgene as a model system to decipher RA function, as
this is the only system in which the location of RA activity can be determined
during development.
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ACKNOWLEDGMENTS |
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