The vertebrate inner ear is a highly intricate sensory organ that
relays two vital sensory inputs to the brain, hearing and
balance. Within the membranous labyrinth of the inner ear, a
relatively small population of cells are directly involved in
sensory transduction. The rest of the labyrinth consists of non-
sensory components that are equally important, and which are
responsible, albeit indirectly, for proper mechanotransduction.
For example, in the vestibular system, the apparatus
responsible for sensing angular acceleration consists of three
non-sensory components: the anterior, posterior and lateral
semicircular canals. Each canal is connected at one end to an
ampulla that houses the sensory tissue – the crista ampullaris
– and at the other end to a non-sensory structure known as the
common crus (Fig. 1). Truncations and size reductions of the
semicircular canals have been shown to result in vestibular
deficits in both mice and zebrafish (Deol, 1983; Ponnio et al.,
2002; Whitfield et al., 1996).
In birds and mammals, the anterior and posterior canals
develop from a common vertical outpouch in the dorsal otocyst
starting at around E3.5 in chicken, whereas the lateral canal
develops from a horizontal outpouch in the middle otocyst. In
the vertical outpouch, the opposing epithelia approach each
other forming two fusion plates that then fuse and resorb,
leaving behind the two tube-shaped canals (anterior and
posterior) connected in the middle by the common crus (Fig.
1) (Bissonnette and Fekete, 1996). Although there are mouse
and zebrafish mutants with defects only in the semicircular
canals, there are no well-characterized mutants in which the
semicircular canals develop normally in the absence of sensory
tissue development (Chang et al., 2004; Anagnostopoulos,
2002; Whitfield et al., 1996). These observations led us to
propose that non-sensory development may require prior
specification by sensory tissues (Cantos et al., 2000).
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).
In the vertebrate inner ear, the ability to detect angular
head movements lies in the three semicircular canals
and their sensory tissues, the cristae. The molecular
mechanisms underlying the formation of the three canals
are largely unknown. Malformations of this vestibular
apparatus found in zebrafish and mice usually involve both
canals and cristae. Although there are examples of mutants
with only defective canals, few mutants have normal canals
without some prior sensory tissue specification, suggesting
that the sensory tissues, cristae, might induce the formation
of their non-sensory components, the semicircular canals.
We fate-mapped the vertical canal pouch in chicken that
gives rise to the anterior and posterior canals, using a
fluorescent, lipophilic dye (DiI), and identified a canal
genesis zone adjacent to each prospective crista that
corresponds to the Bone morphogenetic protein 2 (Bmp2)-
positive domain in the canal pouch. Using retroviruses or
beads to increase Fibroblast Growth Factors (FGFs) for
gain-of-function and beads soaked with the FGF inhibitor
SU5402 for loss-of-function experiments, we show that
FGFs in the crista promote canal development by
upregulating Bmp2. We postulate that FGFs in the cristae
induce a canal genesis zone by inducing/upregulating Bmp2
expression. Ectopic FGF treatments convert some of the
cells in the canal pouch from the prospective common crus
to a canal-like fate. Thus, we provide the first molecular
evidence whereby sensory organs direct the development of
the associated non-sensory components, the semicircular
canals, in vertebrate inner ears.
Supplemental data available online
Key words: FGF2, FGF3, FGF10, Sensory organ, Semicircular
canals, Common crus, BMP2, BMP7
The development of semicircular canals in the inner ear: role of
FGFs in sensory cristae
Weise Chang1, John V. Brigande2,*, Donna M. Fekete2and Doris K. Wu1,†
1National Institute on Deafness and Other Communication Disorders, Rockville, MD 20850, USA
2Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
*Present address: Oregon Hearing Research Center, Oregon Health and Science University, Portland, OR 97239, USA
†Author for correspondence (e-mail: email@example.com)
Accepted 26 May 2004
Development 131, 4201-4211
Published by The Company of Biologists 2004
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
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.
Materials and methods
Fertilized chicken eggs (SPAFAS) were incubated at 38°C, and
embryos were staged according to Hamburger and Hamilton
(Hamburger and Hamilton, 1951). Embryos for paint-fill analysis
were fixed in Bodian’s fixative and processed as described by
Bissonnette and Fekete (Bissonnette and Fekete, 1996).
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.
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 1?108infectious 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).
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-
Development 131 (17)Research article
Fig. 1. A schematic diagram of the chicken inner
ear from E3 to E7. Dotted lines represent the
plane of a cross section through the vertical
canals, shown above each stage. asc, anterior
semicircular canal; cc, common crus; cd, cochlear
duct; ed, endolymphatic duct; fp, fusion plate; psc,
posterior semicircular canal; rd, resorption
domain; vp, vertical canal pouch; A, anterior; D,
4203 FGF signaling in the chicken inner ear
soaked with bovine serum albumin (BSA), or BSA plus heparin
(BSA-heparin) were used as controls and did not result in inner ear
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.
Fate mapping of the canal pouch
We labeled the canal pouch on E4 with DiI at different
locations and observed the fate of labeled cells on E7. The
labeling positions are illustrated in Fig. 2A, with positions 3
and 9 o’clock located dorsal to the presumptive anterior and
posterior cristae, respectively (based on Bmp4 expression), and
the 12 o’clock position at the dorsal tip of the canal pouch. Our
results show that cells labeled between 10 and 2 o’clock
develop solely into the common crus (Fig. 2B-G,K; n=11/11),
with the exception that injections at 12 o’clock sometimes
show additional sporadically labeled cells in the endolymphatic
duct and sac. Injections at 3 and 9 o’clock label primarily the
semicircular canal (Fig. 2H-J,K; n=17/18), and to a lesser
extent the common crus (Fig. 2I,J; n=13/18). Only one out of
18 specimens injected at the 3 and 9 o’clock positions show
DiI labeling in the crista, indicating that we have targeted
successfully regions dorsal to the sensory tissues. Most of the
specimens show a region of canal close to the ampulla that is
devoid of DiI labeling (Fig. 2J, arrowheads), suggesting the
possibility that new cells are continually being generated at the
3 and 9 o’clock positions. Similar labeling patterns are
observed when the same locations are labeled at E5 (Fig. 2L;
n=5). Taken together, these results suggest that there is a canal
genesis zone adjacent to the prospective sensory region that
contributes to the majority of the canal outgrowth.
Bmp2 is expressed in the canal genesis zone
Previously, we have shown that Bmp2 expression is associated
with the outer rim but not the center of the canal pouch at E5,
and that its expression remained associated with the outer rim
Fig. 2. Fate mapping the canal pouch. (A) Otic epithelia were labeled with DiI at the positions shown. Examples are shown at the time of
injection on E4 (B,E,H) and again at E7 (C,D,F,G,I,J,L,M). Partially dissected E7 ears were imaged under fluorescence (C,F,I,L), combined
fluorescence and bright field microscopy (D,G,J) or as a paint-fill for orientation (M). (K) Summary of fate mapping data. The number of
specimens with DiI-labeled cells in the canals and common crus at E7 are shown in red and green, respectively. The common crus (long
arrows), anterior canal (double arrows) and anterior ampulla (short arrows) are indicated. A region between the anterior ampulla and canal
(flanked by arrowheads) is consistently unlabeled. See text for further details. Orientations in B applies to A, E and H. Orientations in D applies
to C,F,G,I,J and L. M, medial; L, lateral; AA, anterior ampulla. Scale bars: in A, 200 µm; in B, 200 µm for E,H; in D, 200 µm for C,F,G,I,J,L.
of the canals after formation (Chang et al., 2002). We now
show, at the earliest stage of detection for Bmp2 transcripts
(E3.5), there are two distinctive wedges of expression along
the anterior and posterior limits of the vertical canal pouch. The
ventral margin of each wedge (Fig. 3A, arrowheads) abuts the
presumptive cristae domains as revealed by Bmp4-
hybridization signals (Fig. 3B,C). These Bmp2-positive regions
correspond to the canal genesis region as indicated by the fate
The prospective common crus domain is Bmp2 and
Bmp2 transcripts are not detectable in the dorsal-central region
of the canal pouch that fate maps to the common crus (Fig. 3A,
arrows). This was confirmed by in situ hybridization using
cryostat sections. The association of Bmp2 expression with the
canals but not the common crus persists well after the canals
are formed at E7; at least up to E12 (Fig. 3D).
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).
Phenotypes elicited by RCAS-Fgf3, RCAS-Fgf10,
FGF2- and FGF10-soaked beads
Both Fgf3 and Fgf10 transcripts are associated with the
neurogenic and sensory regions of the inner ear, similar to what
has been reported in mice (Pirvola et al., 2000) (see Fig. S1 at
http://dev.biologists.org/supplemental/). In addition, two of the
FGF receptors, Fgfr1 and Fgfr2, are weakly expressed in the
canal pouch and highly expressed in the endolymphatic duct
and surrounding otic mesenchyme. To examine the functions
of FGF3 and FGF10 during otocyst development, we
ectopically expressed FGFs in the developing chicken inner ear
by infection with recombinant avian retrovirus encoding Fgf3
(RCAS-Fgf3) or Fgf10 (RCAS-Fgf10), and implantation of
beads soaked with FGF2- or FGF10-heparin.
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;
The phenotypes induced by both RCAS-Fgf3 and RCAS-
Fgf10 are complicated and appear to involve several
developmental processes in the canal pouch, including
resorption and common crus formation. In order to pinpoint the
developmental process(es) and stage(s) that are most sensitive
to FGF treatment, and to explore the downstream targets of
FGFs, we tested the effects of adding FGFs at different stages
of canal development. As FGF3 is not commercially available
and FGF2 has been shown to activate multiple FGF receptors
(Ornitz et al., 1996), the effects of the FGF2 protein on
canal development were tested. Even though weak FGF2
immunostaining was reported in the otic placode and otocysts
(Vendrell et al., 2000), we did not detect any Fgf2 transcripts
in chicken otocysts using in situ hybridization. Nevertheless,
implanting a bead soaked with FGF2 into the lumen of the
inner ear at E4-E5 under standard conditions (see Materials and
methods) interferes with common crus formation by E9 (Fig.
4G, arrows; Table 1, n=40/43). The extent of the common crus
loss is variable, ranging from a complete absence (Fig. 4G,
arrows) to a partial loss of its most dorsal extent (Fig. 6B,
arrows). In the absence of the common crus, the anterior and
posterior canals are continuous with each other, whereas the
lateral canal is often truncated at the location where it normally
joins the common crus (Fig. 4G, arrowhead). Implantation at
an earlier age (E3) using a reduced dose of FGF2 causes a
similar common crus phenotype (n=38/40). A standard dose of
FGF2 at E3, however, causes additional defects in structures
that include the endolymphatic duct and cochlear duct
(n=36/40) that have not been examined in detail. Previous
studies have shown that implantation of FGF2 beads anterior
to the otic placode increased the size of the cochleovestibular
ganglion (Adamska et al., 2001). We did not examine whether
our treatments here, conducted at a later stage of otic
Development 131 (17)Research article
Fig. 3. Expression patterns of Bmp2 and Bmp4 in E3.5 canal
pouches. (A) Arrows indicate the Bmp2-negative domain in the
dorsal region of the canal pouch that will develop into the common
crus. The ventral margins of the two Bmp2 expression domains
(arrowheads in A) abut the Bmp4 sensory domains shown for another
specimen in B. Arrowheads in B indicate Bmp4 expression in the
mesenchyme. (C) Schematic showing the Bmp2 and Bmp4
expression domains superimposed. (D) Horizontal section through
the lateral canal at E9. The Bmp2 hybridization signal is strong in the
thick, outer rim, but weak in the thin, inner rim of the semicircular
canals (arrowheads) and absent in the common crus (arrow).
Orientations in C apply to A and B. Abbreviations are the same as
for Fig. 1. AC, anterior crista; LC, lateral crista; lsc, lateral
semicircular canal; PC, posterior crista. Scale bar in D: 100 µm.
4205 FGF signaling in the chicken inner ear
development, also affect ganglionic development. In the
present study, implantation beyond E5.5 results in normal inner
ears with an intact common crus (n=22/22), suggesting that
only the early stages of common crus specification are sensitive
to excess FGF.
FGF2-bead treatment prevents canal pouch resorption when
assayed on E7, which is prior to the loss of common crus
phenotype first seen at E9 (n=14/15). This lack of resorption
is similar to that seen on E7 following infection with RCAS-
Fgf3 (Fig. 4B). These results suggest that both FGF treatments
affect similar developmental processes, even though the
phenotypes subsequently diverged by E9.
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
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;
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
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
Fig. 4. Ectopic FGF treatments. Paint-
filled inner ears were harvested on E7
(A,B) or E9 (C-J). Embryos were
injected with viruses (A-F) or
implanted with beads (G-J), as
indicated in each panel. Viruses were
delivered to the otocyst lumen on
E2.5-3, except for the ear shown in E,
which received injections into the
periotic mesenchyme on E4. Beads
were implanted on E4 (G,J) or E5
(H,I). White arrows denote defective
regions and arrowheads are described
in detail in the text. Black arrowheads
in D and F indicate normal locations
of the canal pouches. For illustration
of the common crus phenotype, the
endolymphatic duct was removed
from the inner ears shown in E, H and
I. Abbreviations are as in Fig. 1. AA,
anterior ampulla; LA, lateral ampulla;
PA, posterior ampulla. Orientations in
C apply to all panels. Scale bars: in B,
100 µm for A,B; in G, 200 µm for C-J.
Bmp2 expression domain towards the center of the pouch
(presumptive resorption and
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
common crus regions;
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
Development 131 (17)Research article
Fig. 5. Ectopic FGF treatments induce Bmp2 and Bmp7 expression in
the canal pouch. Expression of each gene is indicated on the left of
each panel; treatment group is indicated on the right. Expression is
shown 24 hours after FGF2 bead implantations at E3 (B,D) and E4
(F,H,N), 3 days after RCAS-Fgf3 infection at E4 (J), or 24 hours
after FGF10-heparin bead implantations at E3 (L).
(A-D,K,L) Double arrows indicate differences in Bmp gene
expression in the dorsal canal pouch (presumptive common crus
domain) between treated and control specimens viewed in
wholemounts. (E-H) The approximate level of each section is indicated in either A or B. (E-J) Arrows indicate Bmp2 expression normally
evident in the rim of the prospective canal pouch; arrowheads indicate differences between experiments and controls in Bmp2 expression and
epithelial thickness in the central part of the canal pouch. (M,N) Soho1 expression persists in the central region of the canal pouch (arrowhead)
in both control and treated ears. Orientations in A applies to B-D,K,L; E applies to E-J,M,N. White arrows in B and L indicate the implanted
beads. Scale bars: in E, 100 µm for E,F; in G, 100 µm for G,H; in J, 50 µm for I,J; in N, 50 µm for M,N.
Table 1. Quantitation of FGF-induced phenotypes in the vertical canal pouch
Multiple FGF2 beads†
RCAS-Fgf10 (dorsal mesenchyme)
Age of operation
Age of harvest
Absence of common crus*,¶
Canal pouch non-resorption*
*Number of specimens with the indicated phenotype/total number of specimens scored.
†The common crus is present but narrower than in wild type.
‡Partial loss of the common crus.
§Partial non-resorption of the canal pouch.
¶Numbers represent those specimens that underwent excessive resorption resulting in the loss of the common crus.
4207FGF signaling in the chicken inner ear
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
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
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.
FGFs induce Bmp2 expression before canal pouch
Next, we investigated whether ectopic FGF treatments altered
Bmp2 expression prior to canal pouch formation. We implanted
FGF2-soaked beads into the otocyst at E2.5, before canal
pouch formation, and harvested specimens 18 or 24 hours later
for Bmp2 gene expression analysis. In addition, RCAS-Fgf3
was injected into the mesenchyme surrounding the otic cup at
E2, 12 hours earlier than the bead implantation experiments,
to allow sufficient time for viral integration and transcription,
and then inner ears were harvested at E3 to E3.5. Our results
show that ectopic Bmp2 is increased both by FGF2 beads (Fig.
8A,B; arrowhead; n=12/15) and by RCAS-Fgf3 (Fig. 8C,D;
arrowhead; n=9/14), at an age before the endogenous
expression of Bmp2 is obvious (Fig. 8A,C; arrows). No
significant changes in Bmp2 expression are observed in control
experiments using RCAS-Fgf3-RO (n=16), or BSA beads
(n=6). These results indicate that FGFs are capable of
inducing/upregulating Bmp2 expression at the otocyst stage
before canal pouch formation.
Precocious induction of Bmp7 by FGFs cannot be evaluated
because the otic epithelium is already Bmp7-positive by E2.5,
prior to the initiation of FGF expression (Oh et al., 1996).
Therefore, FGFs in the sensory domain are unlikely to be
required for the induction of Bmp7 expression in the inner ear.
Endogenous FGF activities are required for Bmp2
expression and formation of the semicircular canals
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
Fig. 6. Noggin rescues the loss of common crus induced by FGFs.
Paint-filled inner ears at E9 from (A) controls or (B-D) embryos
implanted at E5 with beads as indicated. Arrows point to the region
of the common crus. Arrowheads indicate truncation of canals.
Asterisk denotes an ectopic epithelial stump. The endolymphatic
ducts in C and D were removed prior to photography. Orientations in
D apply to A-C. Scale bar in C: 200 µm for A,B,D.
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.
The vertical and horizontal canal pouches that develop into the
three semicircular canals are primarily derived from the lateral
wall of the otocyst. In the chicken, fate mapping of the rim of
the otic cup indicates that during otic cup closure, a medial-
lateral lineage boundary is established such that the medial
region of the otocyst develops into the endolymphatic duct and
the lateral region forms the primordial canal pouches (Brigande
et al., 2000). The first physical sign of the endolymphatic duct
and canal pouch primordium as separate entities occurs around
E3.5 (stage 21). However, what drives the continual growth of
either of these structures is not known. Here, we show that a
canal genesis zone located adjacent to the prospective sensory
region is a key component in the continual growth of the canal
pouch (Fig. 9, blue stars). Cells in this genesis zone give rise
to canal epithelium as well as to the common crus. The dorsal
region of the canal pouch (Fig. 9, light blue color) gives rise
to the common crus. Both regions most likely contribute to
cells in the resorption domains.
Bmp2-positive domain and the canal genesis zone
Fate mapping places the canal genesis zone in close proximity
to the Bmp2-positive domain. However, it is not clear whether
both domains completely overlap because a detailed
comparison of the two domains cannot be performed. It is
possible that the two domains do not overlap, and that cells in
the genesis zone are Bmp2 negative and only those that acquire
canal fate become Bmp2 positive. If indeed the two domains
Development 131 (17) Research article
Fig. 7. Fate mapping of the FGF2-treated canal pouch. Otocysts were treated with either BSA (A-C) or FGF2 (D-F) at E4, and DiI was
delivered to the 12 o’clock position of the canal pouch at E5; inner ears were harvested at E9. (G-I) FGF2 bead implantation and DiI injection
were carried out at the same time, at E5. A, D and G show the location of DiI injection. The same three specimens shown in A, D and G are
shown on E9 as fluorescent (B,E,H) or combined fluorescent and bright field images (C,F,I). DiI-labeled cells are associated with the common
crus only in BSA-treated specimens (B,C; arrows), whereas some DiI-labeled cells in FGF2-treated specimens are associated with the canals
(E,F,H,I; double arrows). Arrowheads indicate cells outside the membranous labyrinth and green arrows indicate the junction of the anterior and
the posterior canals. A, anterior; D, dorsal; L, lateral; P, posterior. Scale bar in A: 200 µm for A-I.
4209FGF signaling in the chicken inner ear
overlap completely, then the result of labeled cells found in
both the canals and common crus after injection to the genesis
zone would suggest that some cells migrate out from the Bmp2-
positive domain to form the common crus. Alternatively, these
results can also be explained if the canal genesis zone/Bmp2
domain is relatively small compared with the labeled area
from a single DiI injection. In this case, the chance of
simultaneously labeling cells giving rise to both canal and
common crus is high.
FGFs in sensory tissues promote canal
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
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).
Fig. 8. Effects of FGF and SU5402 treatments prior to canal pouch formation. (A-F) Bmp2 expression in FGF- (B,D) or SU5402-treated (F)
otocysts. A, C and E show left untreated ears of the same embryos that are shown in B, D and F, respectively. Otocysts implanted with an
FGF2-soaked bead at E2.5 (B), infected with RCAS-Fgf3 at E2 (D), or implanted with SU5402-soaked beads at E3 (F) were analyzed 24 hours
later. At E3-E3.5, Bmp2 expression in control inner ears is barely detectable (arrows, A,C), but it is upregulated with FGF2 and RCAS-Fgf3
treatments (white arrowhead in B and D). By E4, the two wedge-shaped Bmp2 expression domains are evident in the control ear (E), and this
expression is downregulated in the SU5402-treated ear (F). The reduction of Bmp2 expression is more pronounced in the posterior domain
(arrowhead) than the anterior domain (arrow). (G-J) Paint-filled inner ears treated with medium (G,I) and high (H,J) doses of SU5402 at E2.5
and harvested at E6 (G,H) or E9 (I,J). (G,I) A medium dose of SU5402 causes the loss of the posterior canal pouch at E6 (asterisk, G) and
prevents formation of the posterior canal and ampulla at E9 (asterisk, I). (H,J) A high dose of SU5402 causes a severely deformed canal pouch
at E6 (arrows, H) and absence of all three canals and ampullae, but the common crus is intact (arrows, J). Arrowhead in J indicates the presence
of the anterior ampulla. Orientations in A apply to C and E; orientations in B apply to D and F; orientations in G apply to H-J.
Ectopic FGF treatments affect common crus
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
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
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
Development 131 (17) Research article
Fig. 9. Model demonstrating how FGFs originating from sensory primordia
regulate semicircular canal and common crus formation in adjacent
epithelium. (A) Progression of canal pouch development from E3.5 to E5.5.
FGF3 and FGF10 emanating from sensory regions (black ovals) promote
canal outgrowth by inducing a canal genesis zone (blue stars), possibly by
activating Bmp2 expression (orange) and, potentially, other factors (X, Y and
Z). Either through physical distance from the sources of FGFs or through
other uncharacterized mechanisms, a FGF-negative, prospective common
crus domain is established (light blue). This prospective common crus
domain is Bmp2 and Bmp7 negative in the dorsal region. Two resorption
domains (light gold ovals surrounded by gold dashes) are established on both
sides of the common crus region in which the epithelia eventually disappear
leaving behind two canals and the common crus (B). (C) A transient,
exogenous dose of FGFs applied before E5 expands the BMPterritory and
prevents the normal resorption process. As the level of exogenous FGF
diminishes over time, the resorption process resumes, and includes the rest of
the epithelia in the common crus domain that failed to be properly specified.
(D) Sustained FGF overexpression by RCAS blocks the resorption process so
that the pouches remain open and a canal-rim fate is adopted.
4211FGF signaling in the chicken inner ear
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.
We are grateful to Drs Susan Sullivan and Tom Friedman for critical
reading of the manuscript, and to Dr Seung-Ha Oh for the construction
of RCAS-Fgf3 used in this study. We also thank Dr Ivor Mason for
mouse Fgf3 cDNA and chicken Fgf3 plasmids, and Dr Sumihare Noji
for Fgf10 cDNA and viral plasmids.
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