1 National Institute on Deafness and Other Communication Disorders, Rockville,
MD 20850, USA
2 Department of Biological Sciences, Purdue University, West Lafayette, IN
47907, USA
Author for correspondence (e-mail:
wud{at}nidcd.nih.gov)
Accepted 26 May 2004
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SUMMARY |
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Key words: FGF2, FGF3, FGF10, Sensory organ, Semicircular canals, Common crus, BMP2, BMP7
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Introduction |
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Analyses of mouse inner ear mutants have identified a number of genes that
are important for the proper formation of the semicircular canals and their
cristae, such as Dlx5, Hmx2, Hmx3 and Fgf10 (for a review,
see Chang et al., 2004). The
role of FGFs in canal development is demonstrated by the loss of all three
semicircular canals and the posterior crista in Fgf10 knockout mice,
and an occasional loss of the posterior canal in one of the reported
Fgf3 knockout mouse lines (Pauley
et al., 2003
; Mansour et al.,
1993
). Identification of the molecular pathways underlying these
phenotypes is complicated by the multiple expression domains of Fgf3
and Fgf10: both genes are expressed in tissues surrounding the otic
placode, as well as in the neurogenic and sensory regions of the otocyst
proper (Pirvola et al., 2000
;
Wright, 2003).
A requirement for Bone Morphogenetic Proteins (BMPs) in canal and crista
development is suggested by manipulating the activities of the proteins in
chicken embryos. Inner ears treated with exogenous Noggin, an antagonist to
BMPs, displayed semicircular canal truncations as well as defective sensory
organs (Chang et al., 1999;
Gerlach et al., 2000
).
However, at least three BMPs are expressed in the chicken otocyst, Bmp2,
Bmp4, and Bmp7 (Chang et al.,
1999
; Oh et al.,
1996
; Wu and Oh,
1996
). It is not clear which BMP(s), or combination of these
proteins, is directly required for the formation of these structures.
Here, we fate map the vertical canal pouch in chicken using DiI. We identify a canal genesis region immediately adjacent to the sensory tissues, which contributes to a majority of the cells in the canals. By delivering exogenous FGFs using beads soaked with FGF2 or FGF10 proteins, or recombinant avian retroviruses encoding Fgf3 or Fgf10, we demonstrate that FGFs in the presumptive cristae promote canal development, most likely by inducing Bmp2 in the canal genesis zone. Ectopic FGF treatments convert some of the cells in the dorsal region of the canal pouch to a canal-like fate.
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Materials and methods |
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Fate mapping
Glass micropipettes (5 µm in diameter) were prepared using a Sutter
Micropipetter Puller P87 and backfilled with a 0.05% solution of Celltracker
CM-DiI (C-7000, Molecular Probes) in 0.3 M sucrose. To visualize the lumenal
side of the otic epithelia, otocysts were first injected with 0.05% Fast Green
in PBS. Then, a small opening was made in an E4 or E5 otic canal pouch at a
location away from the injection site using a tungsten needle. A micropipette
filled with DiI solution was inserted tangentially through the opening into
the otocyst cavity with the aid of a micromanipulator. DiI was then
pressure-injected to the designated area using Pneumatic Picopump PV820 (World
Precision Instrument) under a fluorescent microscope (Leica MZFLIII). Only
embryos without dye leakage into the otic lumen were kept. To further ensure
there was no additional labeling due to possible leakage from the pipette, the
lumen was flushed repeatedly with 0.05% Fast Green solution immediately after
labeling. Each successfully labeled specimen was photographed immediately
after injection, and then again at E7, after harvest and partial
dissection.
In situ hybridization
Whole-mount and in situ hybridization experiments were carried out as
described (Wu and Oh, 1996).
In situ hybridization results presented for each stage are representative of
at least three experiments. Riboprobes for chicken Bmp2, Bmp4, Bmp7
(Chang et al., 2002
),
SOHo-1 (Kiernan,
1997
), Fibroblast growth factor receptor 1-3
(Fgfr) (Walshe and Mason,
2000
), Fgf10 (Ohuchi
et al., 1997
) and Fgf3
(Mahmood et al., 1995
) were
also prepared according to procedures described in the cited references.
Retroviral infection
An avian retrovirus encoding mouse Fgf3 was generated by
subcloning the coding region of mouse Fgf3 (provided by Dr Ivor
Mason, King's College, London) into the ClaI site of an RCAS(A)
vector (Petropoulos and Hughes,
1991). As a negative control, mouse Fgf3 was subcloned in
the reverse orientation (RCAS-Fgf3-RO). The RCASBP(A)-Fgf10
construct containing a 700 bp fragment of the rat Fgf10 cDNA was
obtained from Dr Sumihare Noji (University of Tokushima). Retroviruses were
prepared according to procedures described in Morgan and Fekete
(Morgan and Fekete, 1996
), and
viral stocks with titers of approximately 1x108 infectious
units per ml were used. Viruses were injected into either the lumen of
otocysts or the surrounding mesenchyme as described in the Results section.
The monoclonal anti-gag antibody 3C2 was used to determine the extent of viral
infection (Chang et al.,
1999
).
Bead implantation
Affi-Gel Blue Beads (Bio-Rad) pre-soaked with mouse Noggin-Fc recombinant
fusion protein (R&D Systems) (Chang et
al., 2002), or human recombinant FGF2 (Invitrogen) or FGF10
(R&D Systems) protein, were prepared as described
(Chang et al., 1999
). Briefly,
for our standard treatment, 30 beads were incubated with 1 µl of PBS
containing 1 µg of Noggin, FGF2 or FGF10 plus heparin (10 µg/µl), for
one hour at room temperature and then stored on ice until implantation. For a
standard implantation, a single bead was implanted into an otocyst. The total
number of beads used in the soaking stage for both Noggin and FGFs were
empirically determined, such that a single bead is sufficient to elicit a
canal phenotype after implantation into an E5 otocyst (see Results). To reduce
the amount of protein being delivered in rescue experiments with Noggin in
ovo, the total number of beads used during the soaking stage was increased by
4-fold. To increase the amount of protein being delivered, multiple beads
prepared by the standard method were implanted. Beads pre-soaked with bovine
serum albumin (BSA), or BSA plus heparin (BSA-heparin) were used as controls
and did not result in inner ear abnormalities.
For delivery of SU5402 (Sugen), positively charged AG1 beads (BioRad, AG1-X8) were used. Briefly, 30 AG beads were incubated for 20 minutes at room temperature in 1 µl of 2.5, 5 or 10 mM SU5402 dissolved in DMSO for the delivery of low, medium and high dosages, respectively. After incubation, SU5402-soaked beads were washed three times with sterile PBS, and were stored on ice until implantation. Beads prepared with DMSO alone were used as controls.
FGF- or SU5402-soaked beads were implanted directly into the lumen of the otocyst at the stages indicated, whereas Noggin-soaked beads were implanted into the mesenchyme adjacent to the dorsal region of the vertical canal pouch.
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Results |
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In contrast to Bmp2, Bmp7 expression is not restricted to the
prospective canal rim, and instead becomes elevated in the central region of
the canal pouch by E6 (Chang et al.,
2002). However, similar to Bmp2, Bmp7 is not expressed in
the dorsal region of the canal pouch at E3.5
(Fig. 5C, double arrows). By
E7, after the canals and common crus are formed, Bmp7 is not
expressed in the common crus but in the inner rim of the canals
(Chang et al., 2002
).
|
Injection of RCAS-Fgf3 into the lumen of the otocyst at E2.5-3 (Stages 18-20) results in a failure of canal resorption at E7 when compared with controls (Fig. 4A,B; arrowhead). By E9, the infected inner ears show multiple epithelial protrusions from the non-resorbed canal pouches (Fig. 4D, arrows), although the overall size and relative position of the canal pouches appear normal (Fig. 4C,D; arrowheads; n=15/15). RCAS alone or RCAS-Fgf3-RO did not lead to inner ear defects (n=15). RCAS-Fgf10 yielded a similar phenotype to RCAS-Fgf3, with undulated epithelial outgrowths (Fig. 4F; n=10/16).
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To verify that the two gain-of-function approaches are disrupting the same developmental processes, we conducted two additional experiments in which the effective dosages of FGFs were altered. We increased the dosage of exogenous FGF2 by implanting more FGF2-soaked beads into the lumen of the otocyst at E5, and, as a result, resorption was delayed at least up until E9, although epithelial protrusions similar to RCAS-infected inner ears were not evident (Table 1). By contrast, in an attempt to reduce viral spread, a small dose of RCAS-Fgf3 was injected into the mesenchyme dorsal to the otocyst at E4, 1-1.5 days later than the lumenal injections. A small percentage of these infected inner ears show a milder phenotype on E9, characterized by slightly enlarged semicircular canals (Fig. 4E, asterisk) and a thin common crus (Fig. 4E, arrow; n=4/35 from four separate experiments), thus resembling the bead-implanted inner ears. The phenotypes in the rest of the specimens are similar to the one illustrated in Fig. 4D.
Furthermore, to verify that these phenotypes are elicited by perturbing the
FGF pathway, we simultaneously implanted FGF2 beads and beads soaked with
SU5402, an inhibitor of FGF receptors
(Mohammadi et al., 1997). As
expected, SU5402 is able to prevent the loss of the common crus phenotype
caused by exogenous FGF2 (Fig.
4H,I; arrows; n=5/6).
Fgf10 is expressed endogenously in the developing inner ear prior to canal pouch formation. FGF10-soaked beads have no effect on common crus formation. However, FGF10-heparin beads elicit the loss of the common crus similar to the common crus phenotype induced by FGF2 bead implantation (Fig. 4J, arrows; Table 1, n=5/8). Taken together, these results suggest that a transient presence (bead implantation) or a modest increase (focal mesenchymal RCAS infection) of FGFs during canal pouch development delays the normal resorption process and alters the formation of the common crus. By contrast, prolonged FGF treatment (lumenal RCAS infection) completely blocks resorption and converts the entire canal pouch into a canal duct-like fate.
FGFs induce Bmp2 and Bmp7 in the common crus domain
We next sought to explore downstream effects of the FGFs. Inner ears
implanted with FGF2 beads at E3 to E5 show an induction of Bmp2 and
Bmp7 in the dorsal otocyst within 24 hours, particularly in regions
corresponding to the prospective resorption domains and common crus
(Fig. 5A-D, double arrows).
Although the dorsal epithelium of the canal pouch is normally thin
(Fig. 5E, arrowheads), FGF2
bead implantation causes an induction of Bmp2 expression and an
increase in the thickness of the epithelium that resembles canal-type
epithelium (Fig. 5F,
arrowheads). In a more ventral region of the pouch, where Bmp2
expression is normally restricted to the outer rim
(Fig. 5G, arrows), treatment
with FGF2 expands the Bmp2 expression domain towards the center of
the pouch (presumptive resorption and common crus regions; arrowheads,
Fig. 5G,H). An increase in the
thickness of the otic epithelium is also observed
(Fig. 5H, arrowheads).
Likewise, implantation with FGF10-heparin beads induces Bmp2
expression in the canal pouch (Fig.
5K,L; n=10/11) and increases the thickness of the
epithelium. BSA-soaked beads or BSA-heparin beads do not change Bmp2
expression (n=6).
We verified that these thickened epithelia still retain their canal pouch properties by probing for Soho1, a gene normally expressed throughout the entire canal pouch (Fig. 5M). Despite the change in cellular morphology, Soho1 expression persists in the FGF2-treated ears, suggesting that a conversion to a sensory fate has not occurred (Fig. 5N; n=5).
Noggin rescues the loss of common crus induced by FGF2
To determine whether the induction of Bmp2/7 in the prospective
common crus region is a cause or a consequence of the loss of this structure,
we investigated whether the phenotype could be rescued with a BMP inhibitor.
We implanted beads soaked with Noggin into the mesenchyme surrounding the
dorsal region of the common crus, concurrent with implanting FGF2-soaked beads
to the lumen of inner ears. Noggin rescues the loss of common crus
(Fig. 6C, arrow) but also
results in a partial loss of the semicircular canals
(Fig. 6C, arrowheads;
n=4/4). Presumably, Noggin blocks endogenous BMP activities in the
canal pouch (i.e. canal rim formation), in addition to blocking exogenous BMPs
induced by FGF treatments (i.e. rescue of the common crus). However, by using
a weaker dose of Noggin, the two functions are separable: canal formation is
normal while the common crus phenotype is still rescued
(Fig. 6D; n=5/9). We
conclude that BMP induction by FGF2 is indeed causal to the absence of the
common crus.
Ectopic FGF treatments cause some canal pouch cells to change fate
As the prospective common crus is normally Bmp2 and Bmp7
negative, we used fate-mapping studies to determine whether the loss of the
common crus with FGF2 treatment is possibly due to a change in cell fates. We
implanted chicken otocysts with BSA- or FGF2-soaked beads at E4 and fate
mapped the dorsal rim of canal pouch by injecting DiI into the 12 o'clock
position at E5. By E9, DiI-labeled cells are observed only in the common crus
of the BSA-treated specimens (Fig.
7A-C, arrows; n=6/6), whereas DiI-labeled cells are
incorporated in the canals of specimens treated with FGF2
(Fig. 7D-F, double arrows;
n=5/6). Similar results are observed when the implantation of FGF2
beads is concurrent with DiI labeling at E5
(Fig. 7G-I, double arrows;
n=3/3). Under both treatment conditions, some DiI-labeled cells are
associated with the mesenchyme outside of the labyrinth
(Fig. 7E,F,H,I; arrowheads), a
result not observed with BSA implants (Fig.
7B,C). These results suggest FGF treatments cause some cells in
the dorsal rim of canal pouch that normally give rise to the common crus to
become incorporated into the canals instead.
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Endogenous FGF activities are required for Bmp2 expression and formation of the semicircular canals and ampullae
We used a loss-of-function approach to address whether endogenous FGFs are
required to initiate or maintain Bmp2 expression in the canal pouch.
Otocysts were treated with varying dosages of the FGF inhibitor SU5402 (see
Materials and methods) at E2.5 to E3, i.e. before the initiation of canal
pouch formation, and were assayed after 24 hours for Bmp2 expression.
No change in Bmp2 expression is observed with low doses of SU5402. At
a medium dose, however, the Bmp2 expression domains in the canal
pouch are reduced (Fig. 8E,F,
n=5/6), with the posterior wedge (arrowheads) more affected than the
anterior wedge (arrows). Similar results are obtained when SU5402-soaked beads
are implanted at E5 (n=17/22, data not shown). Control experiments
using DMSO-soaked beads show no reduction of Bmp2 expression
(n=7). These results indicate that Bmp2 expression in the
canal pouch requires FGFs.
To determine the effect of reduced Bmp2 expression on canal formation, SU5402-treated inner ears were paint-filled at E6 and E9. Consistent with the changes observed in Bmp2 expression, a medium dose of SU5402 affects the posterior canal pouch (Fig. 8G, asterisk). Also, the posterior canal (Fig. 8I, asterisk) is more severely affected than the anterior canal (n=20/31). In addition, the posterior ampulla is absent. However, with high doses of SU5402, both the vertical and horizontal canal pouches are affected (Fig. 8H), and all three canals fail to form, although their associated ampullae are sometimes present (arrowhead, Fig. 8J). By contrast, the common crus is intact in all affected specimens analyzed (Fig. 8H,J, arrows; n=20). These results lend further support to the proposal that FGFs promote canal development but are not required for specification of the common crus.
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Discussion |
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FGFs in sensory tissues promote canal development
Using gain-of-function (FGF2, FGF3 and FGF10) as well as loss-of-function
(SU5402) approaches in the developing chicken inner ear, we demonstrated the
requirement of FGFs for canal development. The endogenous sources of FGF3 and
FGF10 are postulated to arise from the neurosensory primordial, and mediate
canal development by inducing Bmp2 in the adjacent canal pouch
(Fig. 9). The restricted
expression of Fgf3 and Fgf10 in the pro-sensory domains, and
the ubiquitous expression of FGF receptors in the otic epithelium, support
this hypothesis.
The significance of BMPs in canal development is supported by our previous
ectopic Noggin treatment studies, even though these studies did not address
which BMP(s) were directly involved (Chang
et al., 1999). The association of Bmp2 in the prospective
canal regions, its upregulation by FGF2, FGF3 and FGF10, and its
downregulation by an FGF inhibitor, all implicate Bmp2. We suggest
that endogenous Bmp2 activity in the canal pouch is regulated by FGFs
associated with the neurosensory primordia. The requirement of Bmp2
in canal development, and its possible interactions with other genes known to
be important for canal development, could be addressed by using Bmp2
conditional-knockout mice, as this FGF-Bmp2 pathway is likely to be
conserved in mice (see below). These experiments are currently underway.
Even though FGFs could also mediate their effects through Bmp7,
the timing of Bmp7 expression tends not to support this; the onset of
Bmp7 expression precedes that of FGFs in the inner ear, suggesting
that Bmp7 induction is not dependent on FGF signaling. Also, the
downregulation of Bmp7 expression in the prospective canal region at
E6 (Chang et al., 2002), when
the canal pouch is still undergoing rapid growth, suggests that Bmp7
may not play a role in maintaining canal development. Finally, unlike
Bmp2, no obvious downregulation of Bmp7 expression was
observed in inner ears treated with SU5402 (data not shown).
Ectopic FGF treatments affect common crus formation
Ectopic FGF treatments also affected formation of the common crus. The
common crus normally forms as a result of resorption of epithelial cells in
the fusion plate, a process that involves programmed cell death in the chicken
(Fekete et al., 1997). It is
not clear whether the formation of the common crus is dictated solely by
regulated resorption of the fusion plate. Alternatively, the prospective
common crus region could be molecularly distinct from the surrounding fusion
plate and could play an active role in regulating the resorption process. This
scenario is supported by the differential Bmp2 expression in the
canal rim but not in the common crus primordia. Other known canal pouch
markers, such as Hmx2, Hmx3, Soho1, Netrin1 and Nor1, do not
distinguish between the two primordial structures
(Fedorov et al., 1998
;
Kiernan et al., 1997
;
Ponnio et al., 2002
;
Salminen et al., 2000
).
The undulation of the otic epithelium, and the absence of resorption in the canal pouch treated with a normal dose of RCAS-Fgf, might be explained by over-proliferation or a lack of programmed cell death (Fig. 4D, Table 1). However, the thinning or lack of the common crus resulting from reduced levels of the RCAS-Fgf or FGF2 bead implantation supports an excess of programmed cell death rather than over-proliferation (Fig. 4E,G; Table 1). Therefore, FGF-induced phenotypes cannot be explained easily by either process. Instead, our fate-mapping data indicate that a cell fate change might be involved. Ectopic FGF2 treatments cause some of the cells in the dorsal rim of the canal pouch, which normally develop into the common crus, to form part of the canals (Figs 2, 7). We hypothesize that cell fate conversion might also derail the normal resorption process. Presumably, as the amount of exogenous FGF in the bead-implanted specimens diminished over time, the resorption process, although resumed, was misregulated and included the common crus domain. Thus, the common crus was absent in FGF2- and FGF10-treated ears (Fig. 9C). This could also explain the extensive DiI labeling in the mesenchyme of FGF-treated specimens at E9 (Fig. 7). However, with sustained FGF expression by viral infection, the resorption was not initiated and the entire canal pouch epithelium adopted a canal-like fate (Fig. 9D). While fate change is one plausible explanation at this point, other as yet unknown mechanisms normally responsible for this epithelial remodeling process might be affected by FGF treatments. Regardless of the mechanisms involved, FGFs can no longer elicit a phenotype beyond E5.5. Furthermore, our results suggest that the prospective common crus plays an active role in regulating the resorption process during normal canal genesis.
It is not clear how FGF concentration is modulated in the dorsal canal
pouch in vivo. So far, expression of Fgfr, or the known FGF
antagonist Sprouty (data not shown)
(Hacohen et al., 1998;
Minowada et al., 1999
), has
not revealed any regional differences in expression patterns that could
account for the low FGF activity in the dorsal region of the canal pouch.
Physical distance from the sources of FGFs could be one plausible
explanation.
Although low levels or absence of FGF activity is required to specify or
maintain a common crus fate, FGF is unlikely to be the only factor required
for this fate. Regulated levels of BMPs are important (see below), and
insensitivity to retinoic acid might also be involved because the common crus
is particularly resilient to retinoic acid treatments
(Choo et al., 1998). In
addition, blocking FGF activity with SU5402 is insufficient to recruit the
surrounding canal pouch epithelium to form an ectopic common crus
(Fig. 3G,H).
Even though Noggin was able to rescue the loss of the common crus by
blocking BMP activities induced by ectopic FGF treatments, the normal
development of the common crus most likely requires regulated levels of BMP
activities rather than a complete absence of BMPs. This is evident by the
absence of a common crus in some of the specimens treated with high levels of
Noggin (Chang et al.,
1999).
Evolutionarily conserved role of FGFs in mediating canal development
In mice, both Fgf3 and Fgf10 are expressed in the
neurogenic and sensory regions of the inner ear
(Pirvola et al., 2000).
Possible functional redundancy of Fgf3 and Fgf10 in the
sensory regions cannot be addressed easily in mice because double knockouts of
Fgf3 and Fgf10 have no inner ear. Presumably, this absence
of otic vesicle formation is due to the lack of earlier FGF3 and FGF10
functions in the hindbrain and mesoderm, respectively
(Alvarez et al., 2003
;
Wright and Mansour, 2003
).
However, canal phenotypes reported in the knockout of either Fgf3 or
Fgf10 support the role of FGFs that is proposed here
(Mansour et al., 1993
;
Pauley et al., 2003
). In
addition, the posterior canal and ampulla are the most affected in
Fgf10 knockout mice, similar to the SU5402-treated specimens in the
chicken. Furthermore, Bmp2 also has a similar spatial and temporal
expression pattern in the canal pouch of mice as in chicken (W.C. and D.K.W.,
unpublished). These results suggest that the role of FGFs in specifying
non-sensory development in the inner ear is most likely evolutionarily
conserved across birds and mammals.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Oregon Hearing Research Center, Oregon Health and Science
University, Portland, OR 97239, USA
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