1 Howard Hughes Medical Institute and Developmental Genetics Program, Skirball
Institute of Biomolecular Medicine, New York University School of Medicine,
540 First Avenue, New York, NY 10016, USA
2 Department of Cell Biology, New York University School of Medicine, 540 First
Avenue, New York, NY 10016, USA
3 Department of Physiology and Neuroscience, New York University School of
Medicine, 540 First Avenue, New York, NY 10016, USA
* Author for correspondence (e-mail: jali{at}uchc.edu)
Accepted 14 January 2005
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SUMMARY |
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Key words: Gbx2, Mouse
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Introduction |
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Acting as cell-intrinsic factors, homeodomain-containing proteins have been
implicated in conferring positional values along the AP and DV axes of the
CNS. In the caudal neural tube, Hox genes play an important role in
establishing and maintaining positional identities of the hindbrain and the
spinal cord (Lumsden and Krumlauf,
1996), whereas two homeobox genes, Otx2 and
Gbx2, are crucial in regulating development of the rostral neural
tube (Joyner et al., 2000
;
Wurst and Bally-Cuif, 2001
).
Otx2 is expressed in the anterior third of the mouse embryo at E7.5,
and is maintained in the forebrain and midbrain at later stages
(Ang et al., 1994
). Correlating
with the later expression, deletion of Otx2 in the neuroepithelium
leads to a loss of the forebrain and the midbrain
(Acampora et al., 1998
;
Rhinn et al., 1998
).
Complimentary to Otx2, the expression domain of Gbx2 extends
from the posterior end of the embryo to the posterior limit of Otx2
at E7.5 (Bouillet et al., 1995
;
Wassarman et al., 1997
).
Gbx2 expression in the neuroepithelium is rapidly downregulated
posterior to r3 after E7.5, and by E8.5 Gbx2 is strongly expressed in
r1, and weakly in r2-3. Brain structures derived from rhombomere 1-3 (r1-3),
including the cerebellum, fail to develop in Gbx2-null mouse mutants
(Wassarman et al., 1997
), and
Otx2 expression appears to be extended to the anterior limit of r4 by
E8.5 (Millet et al., 1999
).
Removal of Otx2 rescues r3 development in Gbx2-null mutants,
demonstrating that Gbx2 plays a permissive role in r3 development by
repressing Otx2 (Li and Joyner,
2001
). Conditional mutagenesis of Gbx2 further
demonstrates that the repression of Otx2 by Gbx2 is required
before E9.0 to allow development of r1-3
(Li et al., 2002
). Although
the genetic evidence has clearly demonstrated an essential role of
Gbx2 for development of r1-3 by repressing Otx2, it remains
unknown whether Gbx2 is sufficient to specify cell fates in r1.
Acting in concert with cell-intrinsic factors, extrinsic factors are
crucial in governing regionalization of the CNS. Embryological and genetic
studies have demonstrated that there is a signaling center (the isthmic
organizer) at the mid/hindbrain junction (isthmus) that plays a central role
in patterning the developing midbrain and cerebellum. Two secreted factors,
Wnt1 and Fgf8, are expressed at the mid/hindbrain junction, and deletion of
Wnt1 or Fgf8 abolishes activity of the isthmic organizer
leading to a loss of all midbrain and r1-derived structures
(Chi et al., 2003;
McMahon and Bradley, 1990
;
Meyers et al., 1998
).
Wnt1 and Fgf8 are normally expressed in two juxtaposed
narrow domains at the Otx2/Gbx2 border with Wnt1 in the
posterior Otx2 expression domain and Fgf8 in the anterior
Gbx2 expression domain. This highly defined spatial expression
pattern of Wnt1 and Fgf8 is dependent on Otx2 and
Gbx2, because in mouse embryos that lack Gbx2 or both
Otx2 and Gbx2, Wnt1 and Fgf8 are expressed in a
broad overlapping domain (Li and Joyner,
2001
; Martinez-Barbera et al.,
2001
; Millet et al.,
1999
; Wassarman et al.,
1997
). The distinct spatial expression patterns of Wnt1
and Fgf8 have been frequently used as an indicator for the normal
formation of the isthmic organizer. However, the regulation and the biological
significance of this spatial expression pattern are not fully understood.
Gain-of-function studies have demonstrated the remarkable inductive
activity of Fgf8 in mimicking the activity of the isthmic organizer
(Crossley et al., 1996;
Liu et al., 2003
;
Martinez et al., 1999
;
Sato et al., 2001
).
Transplantation of beads soaked with Fgf8 recombinant protein in posterior
forebrain or anterior midbrain induce midbrain or cerebellum tissue
(Crossley et al., 1996
;
Martinez et al., 1999
;
Shamim et al., 1999
). However,
expression of Fgf8 in r4 is not able to transform r4 into a
cerebellum in Gbx2-null mutant embryos
(Millet et al., 1999
),
although an isthmus graft is capable of inducing an ectopic cerebellum in the
posterior hindbrain, including r4
(Martinez et al., 1995
). These
observations raise the issue of whether a factor(s), possibly Gbx2,
is missing that is required to mediate Fgf8 signaling to induce a cerebellum
in r4 of Gbx2 mutants.
To investigate the active role of Gbx2 in specifying r1, we
ectopically expressed Gbx2 in r4 using a mouse Hoxb1
enhancer. We show that Gbx2 is not sufficient to induce r1 genes in
r4 in the transgenic mice (HG transgenics). As the Otx2
expression domain expands posteriorly at the late headfold stage in
Gbx2-null mutants, we examined whether a new Otx2/Gbx2
border at the anterior limit of r4 by the five-somite stage can re-establish
an isthmic organizer and partially rescue Gbx2 mutant phenotypes in
Gbx2/ containing the HG transgene.
We show that the normal spatial relationship of Wnt1 and
Fgf8 is restored at the new Otx2/Gbx2 border in
Gbx2/; HG embryos, demonstrating that
juxtaposition of Otx2 and Gbx2 after the five-somite stage
is sufficient to reinstate the spatial expression of Wnt1 and
Fgf8 in Gbx2/ embryos. Despite
co-expression of Fgf8 and Gbx2 in r4, the cerebellum fails
to develop in Gbx2/; HG embryos. Thus,
although Gbx2 is required for development of the cerebellum before
the five-somite stage, other factors are required for mediating Fgf8
signaling during cerebellum development. In
Gbx2/; HG embryos, the expression domain of
Fgf8 is more restricted, whereas it is expanded in conditional
Gbx2 mutants (Gbx2-CKO) in which Gbx2 is removed
after E9.0 (Li et al., 2002).
Interestingly, these two opposite alterations of Fgf8 expression are
both associated with abnormal cell death in the posterior midbrain of
Gbx2/; HG and Gbx2 CKO embryos,
suggesting that cell survival in the posterior midbrain is positively or
negatively regulated by Fgf8, depending on expression levels.
Finally, we show that deletion of Gbx2 disturbs development of r4-6,
and that expression of Gbx2 in r4 of
Gbx2/ embryos rescues development of r3, but
not r1-2 or r4-6, uncovering a new role for Gbx2 in posterior
hindbrain development.
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Materials and methods |
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The r4-Gbx2-Z (7.0 kb) and r4-Gbx2 (8.4 kb) transgenes
were released from the plasmid vectors by SmaI or EcoRV
digest, respectively, and isolated by electrophoresis. These DNA fragments
were further purified by dialysis against microinjection buffer and injected
into mouse zygotes according to standard procedures
(Nagy, 2003).
Histological analysis
Embryos were dissected in PBS and fixed in 4% paraformaldehyde in PBS at
4°C overnight. Embryos were processed for paraffin or frozen sectioning
according to standard procedures (Nagy,
2003). For whole-mount in situ analysis, embryos were dehydrated
and stored in methanol at 20°C. Whole-mount RNA in situ
hybridization was performed based on methods described previously
(Wilkinson, 1992
). Expression
of lacZ in the transgenic embryos was analyzed by X-gal staining
according to established protocols (Nagy,
2003
). RNA in situ hybridization on paraffin or frozen sections
was performed according to methods described previously
(Wassarman et al., 1997
). The
antisense RNA probes were as described previously: Fgf8
(Crossley and Martin, 1995
),
Gata2 (Pata et al.,
1999
), Gbx2 (Bouillet
et al., 1995
), Hoxa2 and Hoxb1
(Wilkinson et al., 1989b
),
kreisler (Mafb Mouse Genome Informatics)
(Cordes and Barsh, 1994
),
Krox20 (Egr2 Mouse Genome Informatics)
(Wilkinson et al., 1989a
),
Otx2 (Ang et al.,
1994
), Spry1
(Minowada et al., 1999
), and
Wnt1 (Parr et al.,
1993
).
Immunohistochemistry
Whole-mount embryo immunostaining with 2H3 antibody supernatant
(Developmental Studies Hybridoma Bank, U. Iowa) was performed as described
(Nagy, 2003).
BrdU cell proliferation assay and TUNEL assay
BrdU cell proliferation assay was performed as previously described
(Mishina et al., 1995).
Pregnant females were intraperitoneally injected with 100 µg BrdU per gram
of body weight 1 hour before they were sacrificed. For the TUNEL assay,
paraffin sections of embryos were dewaxed and apoptosis was detected with
ApopTag (Serologicals, Norcross, GA) according to the manufacturer's
instructions.
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Results |
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To analyze expression of the r4-Gbx2 transgene, we performed RNA in situ hybridization using a Gbx2 cDNA probe. Gbx2 was expressed in r1 and in two longitudinal stripes in the spinal cord of both wild-type and r4-Gbx2 transgenic embryos at E9.5 (Fig. 2B). As predicted, an additional Gbx2 expression domain was detected in r4 of the one r4-Gbx2 transgenic line examined (Fig. 2B). Similarly, ectopic Gbx2 expression was detected in r4 of the four r4-Gbx2-Z transgenic lines by RNA in situ hybridization or by analyzing the activity of ß-galactosidase, which is translated from the bicistronic Gbx2-IRES-lacZ mRNA (Fig. 2C). Two r4-Gbx2-Z transgenic lines (7 and 26) and the one r4-Gbx2 transgenic line (18) were selected for further analysis. The expression level of the transgene appeared from weak to high in the order of line 7, 18 and 26, and the expression level of Gbx2 in r4 was comparable with the endogenous expression in r1 in line 18 (Fig. 2B). In line 26 Gbx2/lacZ was also expressed in the mesenchyme near the isthmus (inset in Fig. 2C). Identical phenotypes were obtained with all three transgenic lines, which we refer to as HG transgenics (for Hoxb1-Gbx2) in the rest of the paper.
Transgenic mice with ectopic expression of Gbx2 in r4 develop normally
Hemizygous HG transgenics were viable and fertile, with no
apparent phenotype. To examine whether ectopic expression of Gbx2 in
r4 interferes with normal r4 development, we analyzed expression of
Hoxb1 and Gata2. Gata2 is normally expressed in the ventral
part of r4 and is a downstream target of Hoxb1
(Pata et al., 1999). The
expression patterns of Hoxb1 and Gata2 were identical
between wild-type and HG transgenic embryos at E9.5 (data not shown).
We then examined whether ectopic expression of Gbx2 in r4 induced
expression of genes that are normally expressed in r1. Fgf8 and
Fgf17 were expressed normally in anterior r1 of HG
transgenic, and no ectopic expression was detected in r4 (data not shown).
Finally, to examine r4 development further in HG transgenics, we
analyzed formation of the cranial nerves using neurofilament immunostaining.
Cranial nerves VII (facial) and VIII (acoustic), which originate from r4, as
well as other cranial nerves formed normally in HG transgenics (see
Fig. S2 in the supplementary material). These data demonstrate that ectopic
expression of Gbx2 in r4 is neither sufficient to transform r4 into
r1 nor to interfere with r4 development.
Expression of Gbx2 in r4 of Gbx2 mutants rescues r3, but not r1, and leads to a loss of the posterior midbrain
To examine whether the HG transgenes can rescue any of the mutant
phenotypes seen in Gbx2 mutants, we introduced the hemizygous
HG transgene insertions onto a Gbx2-null mutant background
(designated as Gbx2/; HG). In both wild-type
and HG transgenic embryos at E10.5, the midbrain and hindbrain are
clearly demarcated by an isthmic constriction, and dorsal r1 is composed of a
bilaterally thickened neuroepithelium, which is the cerebellar primordium
(Fig. 2D,E). In Gbx2
null mutants, the anterior hindbrain, including r1-3 is missing and the
posterior limit of the expanded midbrain is juxtaposed with r4, which is
located immediately anterior to the otic vesicles
(Fig. 2F), as shown previously
(Wassarman et al., 1997). By
contrast, the distance between the posterior limit of the midbrain and the
otic vesicles was significantly increased in
Gbx2/; HG embryos at E10.5
(Fig. 2G), suggesting that the
anterior hindbrain is partially rescued. However, the alar plate of r1 still
appeared to be missing in Gbx2/; HG embryos.
Similar to Gbx2-null mutants, Gbx2/;
HG mutants died at birth. Morphological and histological analysis of
embryos at E12.5 and E18.5 showed that the cerebellum did not form in
Gbx2/; HG mutants, as in
Gbx2/ embryos
(Fig. 3). In contrast to an
expanded midbrain in Gbx2/ embryos
(Wassarman et al., 1997
)
(Fig. 3E,H), the posterior
midbrain (inferior colliculus) was missing in
Gbx2/; HG embryos
(Fig. 3F,I). Therefore,
although expressing Gbx2 in r4 of Gbx2 mutants partially
rescues the anterior hindbrain, it does not rescue the cerebellum and leads to
an additional loss of the posterior midbrain.
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|
There is a duplication of r4 in Gbx2/; HG embryos
As Gbx2 is required to inhibit Otx2 expression in r1-3 at
the late headfold stage and thus allows development of r3
(Li and Joyner, 2001), we
investigated whether the rescue of r3 in Gbx2/;
HG embryos results from repression of Otx2 by the HG
transgenes at the late headfold stage. Expression of the r4-Gbx2-Z
transgene was therefore analyzed by X-gal staining between E7.75 and E8.5. At
the headfold stage, r4-Gbx2-Z expression was found only in the
posterior mesoderm within the primitive streak (data not shown). Expression of
r4-Gbx2-Z in r4 was first detected at the five-somite stage (inset in
Fig. 1F). Furthermore, at this
stage the Otx2 expression was expanded posteriorly and partially
overlapped with Hoxb1 in Gbx2/; HG
embryos by the five-somite stage, similar to that in
Gbx2/ embryos,
(Fig. 1G,F; see Fig. S1E-F in
the supplementary material). These observations demonstrate that the rescue of
r3 seen in Gbx2/; HG embryos by the
eight-somite stage cannot be due to direct repression of Otx2 by
Gbx2 in r3 at the five-somite stage.
To investigate the timing of rescue of r3 by ectopic expression of
Gbx2 in r4, we examined the initiation of Krox20 expression
in Gbx2/; HG embryos. Krox20 is
normally initiated in r3 at the late headfold stage, and by the three-somite
stage Krox20 is strongly expressed in r3 and weakly in r5
(Wilkinson et al., 1989a). In
Gbx2/; HG embryos at the five-somite stage,
although expression of Krox20 was clearly detected in r5, there were
only a few Krox20-positive cells in presumptive r3, posterior to the
expanded Otx2 expression domain
(Fig. 1I). Therefore,
development of r3 is delayed in Gbx2/; HG
embryos.
We further examined development of the anterior hindbrain in Gbx2/; HG embryos at later stages by analyzing the expression of Otx2 and Hoxb1. In Gbx2/ embryos at E10.5, the expression domains of Otx2 and Hoxb1 were largely segregated with a few Otx2-positive cells in Hoxb1 expression domain (Fig. 5B,E). Interestingly, in Gbx2/; HG embryos Hoxb1 was expressed two separate stripes, a narrow band immediately posteriorly to the expression domain of Otx2 and its normal expression domain in r4 (Fig. 5C,F). The rostral transverse band was often discontinuous (Fig. 5F). The expression domains of the HG transgenes, analyzed by RNA in situ hybridization using a Gbx2 cDNA probe or X-gal staining, were identical to those of Hoxb1 in Gbx2/; HG embryos (inset in Fig. 5F; data not shown). Taken together, our marker gene analysis suggests that there is a duplication of r4 in Gbx2/; HG embryos, and that the tissue between the two Hoxb1 expression domains is probably r3-derived tissue rescued by the HG transgenes.
|
Abnormal Fgf signaling in the mid/hindbrain junction region correlates with abnormal cell death in the posterior midbrain of Gbx2/; HG and Gbx2 conditional mutant embryos
An unexpected phenotype in Gbx2/; HG
embryos was the loss of posterior midbrain tissue. To examine the mechanisms
leading to this loss of posterior midbrain in
Gbx2/; HG embryos, we first analyzed cell
proliferation. Using a BrdU-immunohistochemistry assay, we compared cell
proliferation in the posterior midbrain of wild-type,
Gbx2/ and
Gbx2/; HG embryos at E10.5 and E12.5. There
was no obvious difference in cell proliferation among embryos of these
different genotypes (data not shown). We next examined apoptotic cell death
using a TUNEL assay on sections of embryos at E10.5 and E12.5. TUNEL-positive
cells were rarely detected in the mid/hindbrain region in wild-type and
Gbx2/ embryos
(Fig. 6A,B), except in the
dorsal midline region (data not shown). By contrast, in
Gbx2/; HG embryos an increased number of
TUNEL-positive cells was detected in the posterior region of the midbrain at
E10.5 and E12.5 (Fig. 6C; data
not shown). These data indicate that abnormal apoptosis probably contributes
to the deletion of the posterior midbrain in
Gbx2/; HG embryos.
|
We have previously generated a Gbx2 conditional mutant
(Gbx2-CKO), in which Gbx2 is removed between E8.5 to E9.0 in
the midbrain and r1 (Li et al.,
2002). We observed that the midbrain in Gbx2-CKO mutants
was often truncated at the dorsal midline, despite the posterior expansion of
Otx2 into dorsal r1 (data not shown). In addition, the vermis, which
is derived from anterior r1 (Sgaier et al., 2004), is deleted in
Gbx2-CKO mice (Li et al.,
2002
). Therefore, we investigated whether abnormal cell death is
involved in causing these phenotypes. Indeed we found a significant increase
of TUNEL-positive cells in r1 and the posterior midbrain on the parasagittal
sections of E10.5 Gbx2-CKO embryos
(Fig. 6D). Interestingly, in
contrast to Gbx2/; HG embryos, there was an
expansion and increase in the levels of Fgf8 and Spry1
expression in mid/hindbrain junction area of Gbx2 CKO embryos
(Fig. 6L,H). Therefore, an
increase of Fgf8 signaling is associated with increased cell death in the
developing midbrain/r1 region of Gbx2-CKO embryos.
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Discussion |
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Temporal requirements of Gbx2 in cerebellum development and formation of the isthmic organizer
By generating a Gbx2 conditional mutant, we have previously shown
that the lateral cerebellum forms after Gbx2 is removed in r1 at
E9.0, demonstrating a functional requirement for Gbx2 in cerebellum
formation only before E9.0 (Li et al.,
2002). Our current study extends the conditional mutant analysis
by showing that reintroduction of Gbx2 after E8.5 does not rescue
cerebellum development. Thus, Gbx2 function is probably required
earlier than E9.0, at least before the five-somite stage, based on our present
study.
Despite development of a cerebellum, removal of Gbx2 after E9.0
disrupts the normal spatial relationship of the expression domains of
Wnt1 and Fgf8 (Li et
al., 2002). Gbx2 was thus found to have two requirements
in maintaining Wnt1 and Fgf8 expression after E9.0: one in
repressing Wnt1 expression and the other in delineating a narrow
Fgf8 domain (Li and Joyner,
2001
; Li et al.,
2002
). In the present study, we show that the normal spatial
relationship of Wnt1 and Fgf8 is restored by expressing Gbx2
in r4 after E8.5 in Gbx2/; HG embryos,
demonstrating that juxtaposition of Otx2 and Gbx2 after the
five-somite stage is sufficient to establish the normal border of
Wnt1 and Fgf8 expression.
Fgf8 signaling positively and negatively regulates cell survival in the mid/hindbrain region
Although expression of Wnt1 and Fgf8 at the mid/hindbrain
junction has commonly been used as an indicator of an active isthmic
organizer, the functional significance of the spatial relationship of the two
genes and consequences of any small alterations in the levels of expression of
the two genes have not been addressed. In Gbx2-CKO mutants, the
expression domains of Wnt1 and Fgf8 overlap abnormally, and
Fgf8 expression expands posteriorly and is elevated
(Li et al., 2002)
(Fig. 6). We have previously
shown that cell proliferation is reduced within the Fgf8-expressing
cells in both wild-type and Gbx2-CKO embryos
(Li et al., 2002
). In this
study, we have extended our previous analysis by showing that apoptosis is
significantly increased in dorsal regions of the posterior midbrain and
anterior r1 in Gbx2-CKO embryos. We therefore suggest that the loss
of the dorsal posterior midbrain in Gbx2-CKO mutants is due to
abnormal cell death, whereas the loss of the medial cerebellum derived from
anterior r1 results from a combination of reduction of cell proliferation and
an increase in cell death.
In contrast to the overlapping and enhanced expression of Fgf8 and
Wnt1 in Gbx2-CKO embryos, the spatial relationship of
Wnt1 and Fgf8 is restored at the mid/hindbrain junction in
Gbx2/; HG embryos but the two domains are
abnormally restricted. Similar to Fgf8, the expression domains of
Fgf17 and Spry1 are more restricted to the posterior
midbrain of Gbx2/; HG embryos than in
wild-type embryos at E10.5 (Fig.
6; data not shown), suggesting that Fgf8 signaling in
mid/hindbrain junction area is reduced in Gbx2/;
HG embryos. Interestingly, there is a significant increase in the number
of apoptotic cells in the posterior midbrain of
Gbx2/; HG embryos, but no obvious change in
cell proliferation (Fig. 6; data not shown). Our analysis of Gbx2-CKO and
Gbx2/; HG mutants therefore demonstrate that
similar to a paradoxical control of cell survival by Fgf8 in the
developing forebrain (Storm et al.,
2003), Fgf8 signaling regulates cell survival in the mid/hindbrain
region at both high and low levels of expression.
Gbx2 is required for normal development of r4-6
In this study, we have uncovered a new phenotype in the posterior hindbrain
of Gbx2/ mutants. We show that the
transverse stripes of follistatin expression in r1-2 and r4 are missing in
Gbx2/ embryos at E8.5. In addition, the
expression domains of Krox20 in r5 and kreisler in r5-6 of
Gbx2/ embryos at E8.5 are more restricted
than in wild-type embryos, and the lateral-most expression domains of each
gene are mostly missing in Gbx2/ mutants.
These results demonstrate that in addition to the loss of r1-3 in
Gbx2 mutants, development of r4-6 is disturbed. Interestingly,
expression of Gata2 in ventral r4 and Hoxa2 in r5 appears
normal in Gbx2/ mutants
(Fig. 4; data not shown).
Furthermore, formation of motoneurons derived from r4-6
(Wassarman et al., 1997) or
the projections of these neurons analyzed by neurofilament immunolabeling (see
Fig. S2) appears normal in Gbx2/ mutants.
Therefore, the developmental consequence of the abnormal expression of a
subset of genes that specifically mark r4-6 in Gbx2 mutants remains
to be determined.
Gbx2 is initially expressed throughout the posterior embryo in
both the mesoderm and ectoderm at E7.5 and becomes restricted to r1-3 by E8.5.
Interestingly, Gbx2/; HG embryos share the
same r5/6 phenotype as Gbx2/ embryos,
although the HG transgenes are expressed in the posterior mesoderm of
the primitive streak at E7.5. Therefore, the disruption of r5-6 in
Gbx2 mutants is probably due to a loss of Gbx2 in the
ectoderm or mesoderm at the level of r5-6, as the HG transgenes are
not expressed there. Similar to a transient requirement for Gbx2 in
cerebellum development, the transient expression of Gbx2 in r4-6
between E7.5 to E8.5 could be essential for normal development of r4-6.
Alternatively, Gbx2 may act primarily in formation of the isthmic
organizer, which in turn regulates patterning of r4-6
(Irving and Mason, 2000).
Finally, the anomalies in r5-6 could be secondary to abnormal formation of r4
in Gbx2 mutants, as r4 has been shown to function as an organizing
center in zebrafish that regulates development of the adjacent rhombomeres
(Maves et al., 2002
;
Walshe et al., 2002
). In
agreement with this, we observed abnormal expression of follistatin and
Hoxb1 in r4 of Gbx2/ and
Gbx2/; HG mutants.
Ectopic expression of Gbx2 in r4 rescues development of r3 in Gbx2 null mutants
One of the interesting findings in this study is that expression of
Gbx2 in r4 restores expression of Krox20 and Hoxa2
in r3 of Gbx2 mutants, and leads to a duplication of the
Hoxb1 expression domain anterior to the rescued r3. As both
Krox20 and Hoxa2 are expressed in r3 as well as r5, we
examined expression of kreisler, which is specifically expressed in r5-6 at
E8.5. We show that kreisler is only expressed in r5-6, but not in the rescued
rhombomere in Gbx2/; HG embryos. These
results, thus indicate that r3 is rescued in
Gbx2/; HG embryos.
A previous study of Gbx2 mutants showed that an ill-defined region
is present at E10.5 between the midbrain and r4 of Gbx2 mutant
embryos designated as zone X (Wassarman et
al., 1997). In zone X of Gbx2/
embryos, Otx2, Wnt1, Fgf8 and Hoxb1 are abnormally
co-expressed (Wassarman et al.,
1997
) (Figs 1,
5 and
7). We observed that expression
of Otx2 and Hoxb1 overlaps in
Gbx2/ and
Gbx2/; HG embryos by the five-somite stage
(Fig. 1 and see Fig. S1 in the
supplementary material). We speculate that when the Gbx2 cDNA is
driven by the Hoxb1 r4 enhancer in
Gbx2/; HG embryos, Otx2 is
repressed by Gbx2 in the zone X cells that ectopically express Hoxb1.
A stable Otx2/Hoxb1 (Gbx2) border is then established
anterior to zone X in Gbx2/; HG embryos at
the five-somite stage, and subsequently Krox20 is induced in zone X
cells in between the new Otx2/Hoxb1 (Gbx2) border and r4
(see summary in Fig. 7). We
have previously shown that expression of Krox20 and Hoxa2 in
r3 is restored in embryos lacking both Gbx2 and Otx2 after
the eight-somite stage (Li and Joyner,
2001
). However, it is not known whether Krox20 expression
in r3 is initiated normally at the 0- to 1-somite stage or, similar to that in
Gbx2/; HG embryos, is rescued later (at the
5- to 6-somite stage) in Gbx2/;
Otx2/ embryos. Interestingly, we observed a gap
between the expression domains of Otx2 and Hoxb1 transiently
in Gbx2 mutants at the 3- to 4-somite stage (see Fig. S1A,B in the
supplementary material). Therefore, Gbx2 may be essential for the
initiation of Krox20 expression in r3, whereas removal of
Otx2 in zone X in Gbx2-deficient embryos allows regeneration
of r3 in Gbx2/;
Otx2/ embryos or
Gbx2/; HG embryos after the five-somite
stage.
|
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/8/1971/DC1
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