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First published online 13 December 2006
doi: 10.1242/dev.02745
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1 Institute of Neuroscience and Key Laboratory of Neurobiology, Shanghai
Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai
200031, China.
2 Department of Neurology, Ruijin Hospital, Shanghai Jiaotong University School
of Medicine, Shanghai 200025, China.
3 Department of Biochemistry and Molecular Biology, University of Texas, MD
Anderson Cancer Center, Houston, TX 77030, USA.
4 Departments of Anesthesiology, Psychiatry, Molecular Biology and Pharmacology,
School of Medicine Pain Center, Washington University, St Louis, MO 63110,
USA.
Author for correspondence (e-mail:
dingyq{at}ion.ac.cn)
Accepted 15 November 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Lmx1b, Isthmic organizer, Fgf8, Tectum, Cerebellum
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Nakamura et al., 2005;
Wurst and Bally-Cuif,
2001).
Two secreted factors, FGF8 and WNT1, emanated from the isthmic organizer
are required for the development of the midbrain and cerebellum
(Liu and Joyner, 2001b;
Nakamura et al., 2005;
Wurst and Bally-Cuif, 2001).
Beads coated with FGF8 mimic the effects of MHB grafts in chick embryos
regarding the induction of ectopic tectum (dorsal part of the midbrain) and
cerebellum (Crossley and Martin,
1995; Crossley et al.,
1996; Martinez et al.,
1999; Nakamura et al.,
1986). Conversely, inactivation of Fgf8 in the MHB leads
to the loss of the tectum and cerebellum
(Chi et al., 2003;
Meyers et al., 1998;
Reifers et al., 1998). The
domain of Wnt1 expression is located anterior to that of
Fgf8 in the MHB. Unlike Fgf8, Wnt1 does not have induction
activity but is essential for mid/hindbrain development
(McMahon et al., 1992;
McMahon and Bradley, 1990).
Therefore, FGF8 is the key molecule for mediating the induction activity of
the isthmic organizer.
In addition to Fgf8 and Wnt1, transcription factors that
are expressed in the MHB are also involved in mid/hindbrain development.
Otx2 and Gbx2 are among the earliest genes expressed in the
CNS and mark the anterior and posterior epiblast, respectively
(Joyner, 1996;
Joyner et al., 2000;
Liu and Joyner, 2001b;
Nakamura et al., 2005;
Simeone, 2000;
Simeone et al., 2002;
Wurst and Bally-Cuif, 2001).
Within the MHB, Otx2 and Gbx2 act antagonistically to set up
the position of the isthmic organizer, but are not required for the induction
of Fgf8 or Wnt1 expression
(Broccoli et al., 1999;
Li and Joyner, 2001;
Millet et al., 1999;
Rhinn et al., 1998;
Rhinn et al., 1999). The
expression of En1, Pax2 and Wnt1 is earlier, whereas that of
En2 and Pax5 is later than Fgf8 expression in the
MHB (Crossley and Martin, 1995;
Liu and Joyner, 2001b). The
expression of the En1/En2 genes is required for the maintenance but
not for the initiation of Fgf8 or Wnt1 expression
(Liu and Joyner, 2001a;
Thomas and Capecchi, 1990;
Wurst et al., 1994). By
contrast, Pax2 is found to be necessary and sufficient for the
induction of Fgf8 in the MHB (Ye
et al., 2001). However, whether there are other genes involved in
transcriptional control of Fgf8 expression in the isthmic organizer
is unclear.
The LIM homeodomain transcription factor Lmx1b is a mouse
orthologue of chicken Lmx1, which is required for the limb bud
development (Chen et al.,
1998). Its mutation in human causes a dominantly inherited disease
called nail-patella syndrome (Chen et al.,
1998; Morello et al.,
2001). During CNS development, Lmx1b is essential for the
differentiation of midbrain dopaminergic neurons, hindbrain serotonergic
neurons, and medullary and spinal dorsal horn neurons
(Asbreuk et al., 2002;
Cheng et al., 2003;
Ding et al., 2003;
Ding et al., 2004;
Smidt et al., 2000). In
addition to its well known function in neuronal differentiation,
Lmx1b is expressed in the developing MHB in chick and zebrafish, and
misexpression of Lmx1b in the MHB of the chick embryo causes
expansion of the tectum and cerebellum and induces Fgf8 and
Wnt1 expression (Adams et al.,
2000; Matsunaga et al.,
2002), whereas knockdown of lmx1b in the zebrafish
results in the loss of isthmic and cerebellar structures and the loss of
fgf8 and wnt1 expression as well
(O'Hara et al., 2005).
In the present study, we used a loss-of-function approach to analyze the role of Lmx1b in mid/hindbrain development in mice. We focused on the function of Lmx1b in regulating the expression of Fgf8, the essential factor for the induction activity of the isthmic organizer. In addition, we examined the effect of Lmx1b expression on other isthmic organizer-related genes. Our results demonstrate that Lmx1b expression is necessary for the initiation of Fgf8 expression and for the maintenance of several other genes, including Wnt1, En1, En2, Pax2 and Gbx2. Thus, Lmx1b is essential for the initiation and maintenance of the induction activity of the isthmic organizer during mid/hindbrain development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Danielian et al., 1998;
Kimmel et al., 2000; Zhao et
al., 2007). All mouse lines were maintained on a mixed genetic background. To
inactivate Lmx1b in the MHB, we crossed heterozygous Lmx1b
mice with Wnt1-Cre mice, and their
Wnt1-cre,Lmx1b+/- offspring were mated with
Lmx1bflox/flox mice. The desired
Wnt1-cre,Lmx1b-/flox (hereafter referred to as
Lmx1bw CKO) progeny were obtained, and the littermates
(e.g. Wnt1-cre/Lmx1b+/flox, or
Lmx1b-/flox or Lmx1b+/flox) developed
normally; they were used as control embryos. The age of embryos was determined
according to the plug date [considered to be embryonic day (E) 0.5]. Because
the mating time among the mice may differ, the actual developmental stage of
the embryo was further ascertained according to criteria described previously
(Kaufman, 1998). In each set
of experiments, at least three mutant embryos and an equal number of control
embryos were used.
Histological analysis, immunohistochemistry and in situ hybridization assays
Nissl staining and immunocytochemistry were performed as described
(Ding et al., 2004). E18.5
embryos and postnatal day (P) 0 pups were perfused with 4% paraformaldehyde
(PFA) in 0.01 M phosphate buffered saline (PBS; pH 7.4). For Nissl staining
with Cresyl Violet, the brains were embedded in paraffin wax and sectioned at
12 µm in the sagittal plane. For immunostaning, the PFA-fixed brains were
directly sectioned on a cryostat after cryoprotection with 15% sucrose in PBS.
For double staining of calbindin (Sigma) with BRN3b (Santa Cruz), Cy3-labeled
donkey antirabbit IgG (Jackson) and FITC-labeled donkey anti-goat IgG
(Jackson) were used. Rabbit anti-Ki67 antibodies (Novocastra) were employed to
label proliferating cells.
Whole-mount in situ hybridization was performed on embryos fixed in 4% PFA
in PBS, and digoxigenin-labeled ribroprobes were used to detect expression of
Lmx1b (Ding et al.,
2004), En1, En2 (Li
and Joyner, 2001), Fgf8
(Wassarman et al., 1997),
Gbx2 (Li and Joyner,
2001), Hoxa2 (Chi et
al., 2003), Otx2
(Martinez-Barbera et al.,
2001; Suda et al.,
2001), Pax2 (Schwarz
et al., 1999) and Wnt1
(Danielian and McMahon, 1996).
For double in situ hybridization of Lmx1b with Wnt1 or with
Fgf8,the Lmx1b in situ probe was labeled with fluorescein
and visualized in red with Fast Red TR/Napthol AS/MX (Sigma), whereas the
other probes were labeled with digoxigenin and visualized in dark blue with
substrates of BCIP and NBT.
Whole-mount TUNEL
Whole-mount TUNEL was performed as described previously
(Chi et al., 2003) with some
modifications. Embryos were fixed in 4% PFA and stored in methanol, then
rehydrated in PBS containing 0.1% Tween 20 (PBT) and treated with proteinase K
(20 µg/ml in PBT) for 2-8 minutes. The embryos were fixed again with 4% PFA
for 1 hour and permeabilized in a solution of 0.1% sodium citrate and 0.1%
Triton X-100 for 5 minutes on ice. The embryos were incubated with 1xTDT
buffer (Promega) containing 40 µM digoxigenin-dUTP (Roche) at 37°C for
1 hour, then with sheep alkaline phosphatase-labeled anti-digoxigenin
antibodies (Roche), and finally the signals were visualized in alkaline
phosphatase buffer containing NBT and BCIP.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Lmx1b gene deletion leads to defective tectum and cerebellum development
To test the hypotheses that Lmx1b in the MHB regulates the
induction activity of the isthmic organizer, we systematically analyzed the
development of the tectum and cerebellum in the Lmx1b-/-
mutant. At E18.5 and P0, in the absence of Lmx1b it was apparent from
macroscopic examination alone that the tectum and cerebellum were
significantly reduced in size compared with the wild-type
(Fig. 2A,B). This was
corroborated by examination of sagittal brain sections. In the
Lmx1b-/- mutant, the medial portion of the cerebellum was
not visible and the caudal tectum (inferior colliculus) was missing
(Fig. 2B,D). The reduced
cerebellum appeared to fuse with the much reduced superior colliculus
(Fig. 2D), which was further
shown by double immunostaining of BRN3b (POU4F2 - Mouse Genome Informatics)
(Xiang et al., 1996) and
calbindin, markers for the tectum and cerebellum, respectively
(Fig. 2E,F). The cerebellum,
which is located caudal to the inferior colliculus in the control brain
(Fig. 2C,E), became directly
connected with the superior colliculus in the mutant
(Fig. 2F). In addition, the
cerebral cortex and pretetcal region in Lmx1b-/- mutant
mice seemed to extend more caudally compared with the wild-type control
(Fig. 2C,D), presumably as a
result of the reduced tectum (see below).
|
). However,
expression of Brn3a (Pou4f1 - Mouse Genome Informatics), a
marker for the red nucleus in the tegmentum, remained unchanged in the mutant
embryo at E15.5 (Fig. 3P),
although the cerebellar vermis was not seen dorsally
(Fig. 3N). Taken together,
these findings indicate that deletion of Lmx1b causes severe defects
in the development of the tectum and cerebellum.
Lmx1b is required for the initiation of Fgf8 expression
The secreted factor FGF8, which is emanated from the isthmic organizer, is
essential for the development of the midbrain and hindbrain
(Liu and Joyner, 2001b;
Nakamura et al., 2005;
Wurst and Bally-Cuif, 2001).
Therefore, we sought to dissect the possible relationship between the
expression of Lmx1b and that of Fgf8 during mid/hindbrain
development at stages prior to E10.5, the stage at which morphological defects
in the MHB were first observed in the Lmx1b-/- embryo.
In the wild-type embryo, Fgf8 expression was first observed in the presumptive MHB at the 4-somite stage (Fig. 4A). Strikingly, Fgf8 expression was absent in the Lmx1b-/- mutant at this stage (Fig. 4B). By contrast, Fgf8 expression in other regions of the mutant embryo was normal (arrowhead in Fig. 4B), suggesting specific regulation of Fgf8 expression by Lmx1b in the MHB. In the control embryo, Fgf8 expression in the MHB disappeared around E12.5. No Fgf8 expression was detected from the 4-somite stage to E12.5 in the Lmx1b-/- embryo (Fig. 4D and data not shown). Thus, we conclude that Lmx1b expressed in the MHB is required for the initiation of Fgf8 expression in the isthmic organizer.
Lmx1b is required for the maintenance of Wnt1, En1, En2and Pax2 expression
We next examined whether the expression of Wnt1 and other isthmic
organizer-related genes (Joyner,
1996; Joyner et al.,
2000; Simeone et al.,
2002) was also affected by the deletion of Lmx1b. Since
Lmx1b is required for the initiation of Fgf8 expression, and
inactivation of Fgf8 in the MHB causes downregulation and eventual
loss of expression of other genes at the MHB region
(Chi et al., 2003), it is
possible that any change in the gene expression profile in the
Lmx1b-/- embryo may be due to the loss of Fgf8
expression. We first focused on those genes that are expressed in the MHB
prior to Fgf8. In Lmx1b-/- embryos, the
expression of Wnt1, En1 and Pax2 was drastically reduced in
the MHB at the 3-somite stage (Fig.
4F,J,N), whereas they were highly expressed in the wild-type
embryo (Fig. 4E,I,M). The
expression was lost altogether by E9.5
(Fig. 4H,L,P). Because the
reduction in Wnt1, En1 and Pax2 expression occurred before
the absence of Fgf8 in the Lmx1b-/- embryo, the
reduction should have resulted from the inactivation of Lmx1b, rather
than of Fgf8. Furthermore, we found that En2 expression,
which is normally expressed later than Fgf8, was also downregulated
but maintained at a low level until at least E9.5 (data not shown). Thus, the
expression of Wnt1, En1 and Pax2 did not appear to be
causally related to Fgf8 expression, whereas En2 expression
may depend on Fgf8 expression. Nevertheless, it is clear that
Lmx1b expression, although not required for the initiation of
Wnt1, En1, En2 and Pax2 expression, is necessary for their
maintenance in the MHB.
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;
Simeone, 2000;
Simeone et al., 2002;
Wurst and Bally-Cuif, 2001).
We found that Gbx2 expression was normal in
Lmx1b-/- embryos before the 4-somite stage
(Fig. 5B). However, unlike the
wild-type embryo, in which the Gbx2 transcript was found at the
rostral end of the hindbrain (arrow in Fig.
5C), Gbx2 expression was barely detectable in
Lmx1b-/- embryos at the 4-somite stage
(Fig. 5D). By contrast, the
same level of Gbx2 expression was detected in the caudal hindbrain of
both wild-type and Lmx1b-/- embryos (arrowheads in
Fig. 5C,D), suggesting specific
Lmx1b-dependent expression of Gbx2 in the rostral hindbrain.
Since Gbx2 and Otx2 are required for correct positioning of
the isthmic organizer, the loss of Gbx2 in the
Lmx1b-/- embryo may cause a caudal shift of the midbrain
(Simeone, 2000;
Simeone et al., 2002;
Wassarman et al., 1997).
However, we found no change in either the level or the location of
Otx2 expression in the Lmx1b-/- embryos at the
7-somite stage and E9.5 (Fig.
5H,J) as compared with the wild-type
(Fig. 5G,I). The distance
between the two ends of the midbrain (double-arrowed line in
Fig. 5J) remained unchanged,
and the expression pattern of the pretectal marker Pax6
(Walther and Gruss, 1991) was
not affected in the mutant embryo (Fig.
5P), suggesting that the position of the MHB is not changed in
Lmx1b-/- embryos. To further confirm this, we examined
expression of Otx2 and Hoxa2 (a marker for rhombomere 3)
(Chi et al., 2003) in the
mutant embryos. We found no changes in the distance between the two expression
domains up until the 13-somite stage (Fig.
5K-N), at which point increased cell death was detected in the MHB
(see below). Taken together, Otx2 expression is not affected by the
deletion of Lmx1b, and loss of Gbx2 in
Lmx1b-/- embryos does not result in a caudal expansion of
the midbrain.
Abnormal cell death in Lmx1b-/- embryos
The above results showed that in Lmx1b-/- embryos, the
expression of Fgf8 was absent and the expression of many other genes
involved in midbrain/cerebellum development was downregulated. To determine
whether this was caused by abnormal cell death in Lmx1b-/-
embryos, we performed whole-mount TUNEL staining between the 3- and 13-somite
stages, when Lmx1b deletion-induced gene downregulation was
prominent. A small but similar number of TUNEL+ cells were found in
the MHB of both the mutant and wild-type embryos before the 7-somite stage;
this number increased slightly at the 7-somite stage
(Fig. 5R). However, at the
13-somite stage, TUNEL+ cells were markedly increased in the MHB of
Lmx1b-/- embryos, but not in wild-type embryos
(Fig. 5S,T). Thus, the
Lmx1b deletion-induced changes in gene expression cannot be
attributed simply to increased cell death, as this occurred after these
changes in gene expression. On the other hand, because the increased cell
death in Lmx1b-/- embryos occurred after the loss of
Fgf8 expression and the downregulation of several further
developmental genes, it is likely that the abnormal cell death is a
consequence of these changes in gene expression.
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Wnt1-Cre-mediated deletion of Lmx1b in the MHB results in a similar phenotype
As described above, we found that Lmx1b expression in the MHB is
essential for the development of the isthmic organizer. To further confirm
whether specific expression of Lmx1b in the isthmic organizer is
essential, we performed region-specific gene inactivation using a
Wnt1-Cre conditional knockout method. In this
Lmx1bw CKO embryo, there was a slight reduction in
Lmx1b expression in the MHB at the 3-somite stage, but a nearly
complete absence of Lmx1b expression at the 6-somite stage
(Fig. 6A,B). The incomplete
deletion of Lmx1b before the 6-somite stage may be due to the
existence of non-overlapping expression domains of Lmx1b and
Wnt1 in the MHB (Fig.
1). The nearly complete deletion of Lmx1b at the 6-somite
stage may be ascribed to the downregulation of expression of the
isthmus-related genes caused by the inactivation of Lmx1b, and to the
mutual dependence of their expression in the MHB (see Discussion). The
expression of Fgf8 in the MHB was greatly reduced at the 4-somite
stage (data not shown) and was lost altogether at E9.5
(Fig. 6C,D). If Lmx1b
can regulate Fgf8 expression in a dosage-dependent manner, the
remaining Fgf8 expression at the 4-somite stage may be attributed to
the incomplete deletion of Lmx1b in the Lmx1bw
CKO embryo, whereas nearly complete deletion of Lmx1b after the
6-somite stage results in the complete absence of Fgf8
expression.
We also examined the expression of other developmental genes in the Lmx1bw CKO embryos. At E9.5, expression of Wnt1 (Fig. 6E,F), Pax2 (Fig. 6G,H), En1 (Fig. 6I,J) and Gbx2 (Fig. 6M,N) was completely lost, and that of En2 was downregulated (Fig. 6K,L), whereas that of Otx2 (Fig. 6O,P) was unchanged. All these patterns are similar to those found in conventional Lmx1b-/- embryos as described above. Consistent with this, Lmx1bw CKO mice also exhibited a drastic reduction in the size of the tectum and cerebellum (Fig. 7). We thus conclude that the developmental defects in the tectum and cerebellum are due to the specific action of Lmx1b in the MHB.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Nakamura et al., 2005;
Wurst and Bally-Cuif, 2001).
The molecular basis of the isthmic organizer patterning is only partially
characterized. The growth factor FGF8 has been shown to be responsible for the
induction activity of the isthmic organizer. In the present study, we showed
that expression of Lmx1b precedes that of Fgf8
(Fig. 1), and Fgf8
expression was absent in the MHB of Lmx1b-/- embryos
(Fig. 4). Importantly, a
previous study has shown that misexpression of Lmx1b in chick embryos
induces ectopic expression of Fgf8 and causes expansion of the tectum
and cerebellum (Matsunaga et al.,
2002), suggesting that Lmx1b is sufficient for the
initiation of Fgf8 expression. Conversely, knockdown of
lmx1b in zebrafish results in the loss of fgf8 expression
(O'Hara et al., 2005). Our
results provide the first evidence that Lmx1b is required for the
initiation of Fgf8 expression in the isthmic organizer of mouse
embryos. Together with previous studies, we conclude that Lmx1b is
necessary and sufficient for the initiation of Fgf8 expression in the
MHB during isthmic organizer development, although the intrinsic molecular
machinery may be different among the different species. Furthermore, it is
interesting to note that misexpression of Fgf8 in the chick embryo
(Matsunaga et al., 2002), or
addition of FGF8-coated beads to mouse midbrain slice culture
(Liu and Joyner, 2001a), is
able to induce ectopic Lmx1b expression. Thus, Lmx1b and
Fgf8 appear to cross-regulate each other during the development of
the isthmic organizer. Thus, such cross-regulation between Lmx1b and
Fgf8 may come into play only at the 4-somite stage when Fgf8
expression initiates. Because Lmx1b expression precedes Fgf8,
Lmx1b is likely to regulate expression of Fgf8, either directly
or indirectly, and a positive feedback regulation between Fgf8 and
Lmx1b may exist afterwards to ensure the proper activity of the
isthmic organizer.
Lmx1b is required for maintaining Wnt1 expression
The secreted factor Wnt1 is necessary for the proper development
of the isthmic organizer and mice lacking Wnt1 show abnormal
development of the midbrain and hindbrain
(McMahon and Bradley, 1990;
McMahon et al., 1992). In the
Lmx1b-/- embryos, Wnt1 was downregulated in the
MHB and prematurely lost by E9.5 (Fig.
4). This result indicates that Lmx1b is required for the
maintenance of Wnt1 expression in the MHB. This is consistent with
the previous findings that misexpression of Lmx1b in chick embryos
induces Wnt1 expression ectopically
(Adams et al., 2000;
Matsunaga et al., 2002), and
that knockdown of lmx1b in zebrafish leads to the loss wnt1
expression (O'Hara et al.,
2005). Whether Wnt1 is directly regulated by
Lmx1b is unclear. Because Fgf8 is required for maintaining
Wnt1 expression in the MHB (Chi et
al., 2003; Reifers et al.,
1998), and Fgf8 is absent in the MHB of the
Lmx1b-/- embryo, it is possible that the Fgf8
activity mediates the maintenance of Wnt1 expression by
Lmx1b in the MHB. However, as downregulation of Wnt1
occurred prior to the 4-somite stage, our data revealed an
Fgf8-independent but Lmx1b-dependent Wnt1
expression profile in the development of the MHB prior to the 4-somite stage.
Furthermore, although ectoptically expressed Wnt1 can induce
Fgf8 expression in the chick embryo
(Ye et al., 2001), it is
unlikely that the absence of Fgf8 expression in the
Lmx1b-/- embryo is due to downregulation of Wnt1,
because the initiation of Fgf8 expression occurs independently of
Wnt1 (Danielian and McMahon,
1996; McMahon et al.,
1992). Taken together, Lmx1b is essential for the
initiation of Fgf8 expression and for the maintenance of
Wnt1 expression during development of the isthmic organizer.
IsLmx1b required for the positioning of the MHB?
It is well established that Otx2 and Gbx2 are required
for the correct positioning of the isthmic organizer, and are not required for
the initiation of Fgf8 or Wnt1 expression
(Joyner et al., 2000;
Martinez-Barbera et al., 2001;
Rhinn et al., 1998;
Simeone, 2000;
Simeone et al., 2002;
Wurst and Bally-Cuif, 2001).
Expression of Lmx1b precedes Gbx2, but after Otx2
(Fig. 1). In
Lmx1b-/- embryos, Gbx2 expression was normal
before the 4-somite stage, but became barely detectable at the 4-somite stage
and absent thereafter (Fig. 4).
By contrast, Otx2 expression in Lmxb-/- embryos
was similar to that in the wild-type control (Figs
3,
4 and
5). This is interesting because
Lmx1b appears to differentially regulate the expression of
Gbx2 and Otx2, which are known to antagonize each other in
setting the position of the MHB. These results also argue for the specificity
of Lmx1b-dependent regulation of gene expression in the MHB.
Moreover, Gbx2-/- mice lack the anterior hindbrain and
display abnormal caudal expansion of the midbrain, paralleled by posteriorized
expression of Otx2, Wnt1 and Fgf8
(Broccoli et al., 1999;
Li and Joyner, 2001;
Wassarman et al., 1997).
However, we did not find such a caudal shift of the midbrain when
Gbx2 expression was lost in the Lmx1b-/- embryo,
as indicated by the normal expression of Otx2 and Pax6 and
unchanged distance between the Otx2+ and
Hoxa2+ domains in the mutant embryo
(Fig. 5). The reduction in this
distance in the mutant embryo at the 13-somite stage is most likely to be due
to the increased cell death in the MHB, rather than to caudal extension of the
midbrain (Fig. 5). This raises
the possibility that Lmx1b deletion in the MHB may activate an
unknown compensatory mechanism that prevents caudal shift of the midbrain in
the absence of Gbx2 activity. It is also possible that Lmx1b
may be required cell-autonomously in the putative expanded domain for
Otx2 expression in the MHB.
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|
; Nakamura et
al., 2005; Wurst and
Bally-Cuif, 2001; Ye et al.,
2001). We have shown that the expression of Lmx1b
precedes that of Pax2 and En1
(Fig. 1), and that
Lmx1b deletion downregulated their expression
(Fig. 4). Because the
downregulation of these transcription factors occurred at a time prior to the
initiation of Fgf8 expression in the normal embryo, it is unlikely
that this downregulation is caused by the loss of FGF8 activity in the mutant
embryo. This does not exclude the possibility that Fgf8 normally may
contribute to the expression of these transcription factors at later stages,
because Fgf8 is required for their maintenance
(Chi et al., 2003).
A positive maintenance loop involving Fgf8, Wnt1 and En
and Pax genes during the development of the isthmic organizer has
been proposed (Joyner et al.,
2000; Nakamura et al.,
2005; Wurst and Bally-Cuif,
2001). This is based on the evidence that the loss of En1,
Pax2, Fgf8 or Wnt1 function affects the maintenance but not the
initiation of expression of these genes, with the exception of Pax2
that is required for the initiation of Fgf8 expression
(Chi et al., 2003;
Joyner et al., 2000;
McMahon et al., 1992;
Meyers et al., 1998;
Wurst et al., 1994;
Ye et al., 2001). Our study
indicates that Lmx1b is a crucial component of the positive
maintenance loop for isthmic organizer development. Lmx1b is required
not only for maintaining Wnt1 and Pax2 expression, but also
for the initiation of Fgf8 expression. Because the downregulation of
Wnt1 and Pax2 may not be sufficient to explain the loss of
Fgf8, Lmx1b may control the initiation of Fgf8 either
directly or via an unknown pathway, which may in turn regulate expression of
Lmx1b. The complexity of this positive maintenance loop remains to be
fully elucidated. For example, we have found that Lmx1b expression is
lost in En1-/- embryos at E9.5 (C.G. and Y.-Q.D.,
unpublished). Since Lmx1b is normally expressed earlier than
En1, the immediate maintenance of Lmx1b may require possible
feedback regulation by En1.
In summary, there are two steps in the regionalization of the developing
mid/hindbrain (Adams et al.,
2000; Beddington and Robertson,
1998). The first step is the positioning of the future MHB in the
early embryo, for which Otx2 and Gbx2 are two major players;
this process is Lmx1b-independent because in the
Lmx1b-/- embryo there was no caudal shift of the
Otx2+ domain, which marks the MHB. The second step of the
regionalization begins when the isthmic organizer is established in the MHB.
We propose that, as a key component of the genetic pathway underlying the
isthmic organizer activity, Lmx1b is responsible for the initiation
of Fgf8 expression and the maintenance of other key isthmic organizer
genes.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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