BACKGROUND
the inner ear is derived from a thickened patch of ectodermal cells, the otic placode, which develops lateral to the developing hindbrain.
in birds and mammals, the otic placode invaginates and pinches off to form the pear-shaped otic vesicle, or otocyst.
the inner ear's sensory epithelia originate from the ventromedial region of the otocyst, an area that can be defined by the expression of several markers, such as ben  <cit> , lunatic fringe and cserrate- <dig>  <cit> , islet- <dig>  <cit>  and bone morphogentic proteins   <cit> .
the distinctive temporal and spatial expression patterns of bmp <dig> in almost all developing chicken inner ear sensory epithelia has led to the suggestion that this protein may play an important role in the induction of inner ear sensory organs  <cit> .
because homozygous bmp <dig> knockout mice die between e <dig>  and e <dig>   <cit> , a period before the inner ear has formed, it is difficult to analyze the role of bmp <dig> in the genesis of inner ear sensory organs.
as a consequence, other model systems, such as the chicken embryo, have been used to study possible roles of bmp <dig> in the inner ear.
for example, noggin, an antagonist of bmps, has been employed to interfere with bmp signaling during chicken inner ear development resulting in defects in semicircular canal formation and otic capsule malformation  <cit> .
although malformed or missing cristae were observed in these experiments, the hair cells developed normally.
thus these results did not clarify why bmp <dig> is robustly expressed in sensory epithelia primordia thereby neither confirming nor refuting the hypothesis that bmp signaling is involved in the genesis of inner ear sensory organs.
a possible explanation for the previously observed lack of sensory epithelia defects after in ovo application of bmp <dig> antagonists is that the antagonists did not penetrate far enough to reach sufficiently high concentrations in the developing sensory epithelia to block bmp signaling effectively.
to address this issue, we exploited a serum-free floating otocyst culture system, which allowed us to quantitatively analyze progenitor cell proliferation, apoptosis and cell differentiation in the developing otocyst with loss of function and gain of function experiments.
our experiments revealed that bmp <dig> signaling is involved in generation of sensory epithelia by negatively regulating inner ear progenitor cell proliferation through downregulation of the homeodomain transcription factor pax- <dig>  subsequently, bmp <dig> signaling promotes hair cell differentiation.
RESULTS
exogenous bmp <dig> leads to increase in hair cell numbers and blockade of bmp signaling inhibits hair cell generation
maintenance of the developing avian inner ear in vitro in an environment with largely reduced extrinsic influences can be facilitated by removal of periotic mesenchyme, which otherwise could serve as source for various signaling molecules  <cit> .
as much as possible, we assured removal of the mesenchymal tissue surrounding the otocyst by mild enzymatic treatment with trypsin and careful dissection.
we found that removal of the periotic mesenchyme did not affect the capability of stage  <dig> otic vesicles to generate major inner ear cell types after 7d in serum-free suspension culture .
vesicles with obvious mesenchymal contributions that gave rise to cells with chondrocyte morphology  were excluded from this study.
only otic vesicles that did not display any obvious sign of mesenchymal contribution after culture were included in this study .
although free-floating otocysts did not differentiate into morphologically well-defined and structured inner ears, they displayed remarkable constancy with regard to the number of total cells after  <dig> days in culture  and of the number of hair cells formed during the culture period .
we identified hair cells with antibody to myosinviia  <cit>  and by their hair bundles labeled with antibody to the hair bundle markers espin and hair cell antigen  <cit>  .
furthermore, the hair cells that differentiated in free-floating otocysts appeared to be contacted by neurites, identified with antibody to neuron-specific β-iii tubulin .
to assess the effects of bmp signaling on otocyst cells and hair cell generation, we analyzed complete sets of serial sections obtained from individual specimens of each experimental group.
when exogenous bmp <dig> was added on culture day three, we observed a substantial increase in the number of hair cells, when compared with the untreated controls ; hair cells were identified by double-immunostaining for myosinviia and hair cell antigen.
blocking of bmp signaling with either noggin or soluble bmp receptor 1a or 1b proteins dramatically reduced hair cell numbers after seven days in culture .
the effect of noggin on hair cell generation was dosage-dependant and could be rescued by adding exogenous bmp <dig> .
exposing cultures to different concentrations of bmp <dig> revealed an optimal concentration of 3– <dig> ng/ml, which yielded the greatest number of hair cells .
bmp <dig> inhibits otocyst cell proliferation and induces apoptosis in prospective inner ear ganglion cells
to elucidate the effect of bmp <dig> on the increased generation of hair cells, we analyzed cell proliferation and apoptosis in cultures treated with exogenous bmp <dig> from day three in culture onward.
we found that proliferation was significantly reduced in otocysts treated with bmp <dig> whereas apoptosis was increased .
bmp4-induced apoptosis almost exclusively occurred in areas that we identified as neuronal using the strong islet- <dig> immunostaining of cochleovestibular neuron nuclei .
islet- <dig> is expressed in the developing inner ear in neurons as well as in the nascent sensory epithelia; neuronal islet- <dig> staining can easily be distinguished from islet-1-positive developing sensory epithelia by a more round shape of stained neuronal nuclei and higher intensity when compared to epithelial staining .
the paired-box transcription factor pax- <dig> is expressed in developing sensory epithelia and is down-regulated in cochleovestibular ganglion cells  <cit>  and accordingly, neuronal domains in cultured otocysts lack pax-2-expression .
when bmp signaling was blocked with noggin, there was no significant increase in overall cell proliferation, but less apoptosis occurred in the neuronal area, identified by staining for β-iii tubulin .
based on these results, we rule out that the increase in hair cell numbers in response to bmp <dig> is based on a higher rate of cell proliferation or lower rate of apoptosis.
otocyst cells fail to commit to a sensory epithelial fate in the absence of bmp signaling
the sensory epithelia of the inner ear, including hair cells and supporting cells, are derived from pax-2-positive progenitor cells that also give rise to other inner ear cell types  <cit> .
the reduction in the number of hair cells in response to blockade of bmp signaling with noggin was not accompanied by a detectable overall reduction of pax-2-positive progenitor cells .
as the sensory epithelium progenitor cells become committed to form sensory patches, pax- <dig> expression decreases and islet- <dig> becomes detectable in both nascent hair cells and supporting cells before the onset of expression of hair cell markers  <cit> .
we found that in noggin-treated otocysts, islet-1-positive epithelial patches containing hair cells appeared noticeably diminished, whereas the more strongly islet-expressing cochleovestibular neurons were apparently not affected .
these data support our hypothesis that the generation of sensory patches from pax-2-expressing progenitors depends on bmp signaling.
bmp <dig> inhibits cell proliferation through downregulation of pax-2
the expression of pax- <dig> in the early developing inner ear is usually correlated with increased cell proliferation and absence of apoptosis, which mostly occurs outside of the pax-2-positive regions  <cit> .
when bmp <dig> was added at the beginning of the 7d culture period, we noticed a substantial decrease in the number of pax-2-positive cells, although without an apparent effect on the developing sensory epithelium .
western blot analysis corroborated that bmp <dig> down-regulates pax- <dig> expression in a dose-dependent manner .
in the normal inner ear, bmp <dig> mrna is used as a marker to visualize presumptive sensory epithelium  <cit> .
at the crucial time during development, when sensory epithelia become postmitotic, epithelial islet- <dig> expression overlaps with bmp4-expressing areas .
conversely, in the same presumptive sensory epithelia, pax- <dig> expression is downregulated , suggesting that bmp <dig> acts as suppressor of pax- <dig> expression during normal inner sensory epithelium formation.
discussion
bmp signaling is required for inner ear sensory organ formation
the discovery of bmp <dig> mrna expression in all sensory organ primordia of the developing inner ear has led to the hypothesis that this signaling protein plays a role in the induction or differentiation of inner ear sensory epithelia  <cit> .
however, because homozygous bmp <dig> knockout mice do not live beyond the ninth day of embryonic development it is not feasible to analyze bmp <dig> function during murine otogenesis.
to circumvent this problem, we used a serum-free floating culture technique, which allowed us to quantitatively test the function of bmp <dig> on avian inner ear sensory epithelium formation in a controlled environment.
this culture regimen enabled us to quantitatively analyze the generation of inner ear cell types by using cell-specific markers.
we found that the number of hair cells that formed in otocysts cultured for  <dig> days is considerably lower than the number of hair cells that can be found in a chicken ear at the 10th day of embryonic development .
nevertheless, we argue that the chicken inner ear at e <dig> is also substantially larger than the 7-day cultured otocyst, which makes it difficult to assess whether a ratio of ~ <dig> % hair cells of the total cell number in a floating otocyst is comparable to the ratio of hair cells versus total inner ear cell number at e <dig>  nevertheless, we suggest that that the effects we observed in vitro are at least partially representative of the hair cell populations found in the various hair cell-bearing organs of the developing chicken ear.
to the best of our ability, we removed periotic mesenchyme from the e <dig> otocysts.
specimens with obvious mesenchymal contribution were not scored in this study.
nevertheless, we did not observe a noticeable difference in the number of hair cells and neurons detectable in otocysts with clear mesenchymal contribution, which is an indication that the periotic mesenchyme appears to exert little influence on the otocyst after stage  <dig> .
loss of bmp signaling by application of noggin or a combination of dominant-negative bmp receptors markedly reduced the number of hair cells in the developing otocyst, which strongly supports the hypothesis that bmp signaling is required for hair cell generation.
nevertheless, even at the highest concentrations of noggin or dominant-negative bmp receptors, no complete blockade of hair cell generation was achieved.
we suspect that the nature of bmp signaling in the developing sensory epithelia does not allow complete blockade with application of soluble inhibitors.
bmp <dig> is a secreted protein that binds to cell surface receptors to activate the smad signaling pathway .
secreted bmp <dig> acts on neighboring cells in a paracrine manner as well as in autocrine fashion on the cells that produce the factor if these cells have appropriate bmp receptors.
we hypothesize that extracellular bmp inhibitors cannot completely interfere with autocrine signaling, resulting in an incomplete blockade of bmp signaling in otocyst cells that express both bmp <dig> and bmp receptors.
interference with bmp signaling in the inner ear in vivo by application of noggin or noggin-producing cells  <cit>  led to inhibition or malformation of semicircular canals accompanied by occasional deformation of ampullae.
no effects on sensory epithelium formation and hair cells were reported; however, these studies did not explicitly quantify the number of hair cells.
alternatively, we hypothesize that dorso-lateral transplantation of beads soaked with noggin or of noggin-producing cells into the periotic mesenchyme apparently did not suffice to reach sufficient concentrations of the inhibitor over the course of several days in the ventro-medial region of the otocyst, where the sensory epithelia originate.
using a serum-free floating culture environment allowed us to control the concentration of bmp <dig> or bmp signaling inhibitors more precisely than previous in ovo experiments.
bmp <dig> promotes hair cell differentiation
three mechanisms could account for increase of the number of hair cells by exogenous bmp4: 1) promotion of sensory epithelial progenitor cell proliferation, 2) prevention of progenitor cell apoptosis, or 3) promotion of progenitor cell differentiation to hair cells.
our results suggest that bmp <dig> inhibits otocyst cell proliferation, but affects apoptosis only in the neuronal domain of cultured otocycts, implying that a plausible mechanism of bmp4's effect on increasing the number of hair cells is to promote sensory epithelium progenitor cells to differentiate.
another line of evidence for bmp4's effect on sensory epithelium progenitor cells arises from our loss-of-function analysis with noggin.
here we found that noggin-treatment did not affect the number of pax-2-positive cells, whereas the number of hair cells was considerably reduced.
this result implies that pax-2-positive progenitors fail to differentiate into sensory epithelium when bmp signaling is blocked.
our observations that bmp4-treatment leads to increased hair cell generation and to reduction of otocysts cell proliferation, suggests a dual, concentration-dependant, action of bmp4: at concentrations of 3– <dig> ng/ml bmp <dig> augments cell differentiation.
in fact, we did not find a significant inhibition of cell proliferation with bmp <dig> concentrations below  <dig> ng/ml .
at higher bmp <dig> concentrations , cell proliferation is sufficiently reduced to offset the bmp <dig> effect on hair cell differentiation.
the result of the concentration-dependant dual effects of bmp <dig> is manifested in a bell-shaped dose-dependency of hair cell numbers .
the effects of bmp <dig> on otocyst cell proliferation and apoptosis that we report here differ from, but do not contradict, results obtained by in vivo administration of noggin to the developing chicken inner ear  <cit> .
in these experiments, cell proliferation decreased and apoptosis increased after noggin-secreting beads or cells were transplanted into the mesenchyme adjacent to the otocysts.
we argue that these previous analyses and our experimental protocol are not easily comparable as many factors could account for the observed differences.
for example, interactions of noggin with potential signals in the periotic mesenchyme might produce different effects on inner ear tissue when compared with mesenchyme-free otocyst cultures.
another possible explanation is that our analysis included all otocyst-derived cells, whereas previous studies focused on the parts of the developing inner ear surrounding the noggin-secreting grafts.
the effect of bmp <dig> on promoting differentiation of sensory epithelium progenitor cells is consistent with its temporal and spatial expression in the inner ear sensory organ development  <cit> .
bmp <dig> mrna is expressed in all sensory organ primordia before the hair cells and supporting cells are developed.
at this crucial period of sensory organ formation, the progenitor cells downregulate pax- <dig>  leave the cell cycle, and initiate differentiation into hair cells and supporting cells; pax- <dig> expression subsequently persists in hair cells  <cit> .
these events appear to be correlated with the presence of bmp <dig> in sensory patches, supporting a possible role for bmp <dig> in controlling proliferation and specifying differentiation in the inner ear's sensory epithelia.
additional clues about the potential targets of bmp <dig> in the developing inner ear could arise from analysis of expression of bmp receptors and components of the bmp signaling pathways.
beside a comprehensive study in the developing zebrafish ear and lateral line  <cit> , only one study has addressed in the developing chicken inner ear expression of bmp receptors and smad proteins, the intracellular transducers of bmp signaling  <cit> .
although the latter study was focused on the role of bmp signaling during development of semicircular canals, it should be noted that the expression of bmp receptors in the developing chicken inner ear is probably not restricted to sensory patches or the pax-2-positive domain.
a more widespread expression of bmp receptors could potentially be the reason for the substantial downregulation of pax- <dig> protein that we observed in free-floating otocycts in response to bmp <dig> treatment.
at least in the developing zebrafish inner ear several bmp receptors are expressed widely, some of them ubiquitously, at all developmental stages  <cit> .
bmp <dig> is a crucial control factor of proliferation, differentiation and apoptosis in the developing inner ear
at early stages of inner ear development, all cells of the otic placode and the otic pit express the transcriptional regulator pax- <dig>  subsequently and noticeably evident at embryonic day three, pax-2-expression gets more localized to the medio-ventral part of the otic vesicle, the region where the sensory epithelia originate  <cit> .
at this time of development, cell proliferation mostly occurs in the pax- <dig> expression domain and apoptosis happens outside of the pax- <dig> positive domain  <cit> .
we show that the numbers of pax-2-positive cells as well as proliferating cells are markedly reduced when exogenous bmp <dig> is added to cultured otocysts.
coinciding with decreasing numbers of pax-2-positive cells, we also detected substantial downregulation of the expression of pax- <dig> protein.
in the developing sensory patches, we were able to visualize that bmp <dig> mrna is detectable in the incipient sensory patches just at the time when islet- <dig> expression becomes evident.
the onset of islet- <dig> expression in sensory patches coincides with early differentiation of sensory epithelial cells  <cit> .
in parallel, pax- <dig> protein is markedly downregulated in these patches, implying that bmp <dig> also causes downregulation of pax- <dig>  cessation of proliferation, and upregulation of markers for early differentiating sensory epithelium in vivo.
CONCLUSIONS
in summary, our results indicate that bmp signaling is involved in the generation of inner ear sensory patches and hair cells.
we also found evidence that bmp <dig> can affect cell proliferation and apoptosis in different cell populations of the developing inner ear.
we therefore conclude that correct control of bmp signaling in all areas of the developing otocyst is central to obtaining an accurate balance of cell proliferation, apoptosis and cell differentiation needed for correct morphogenesis of the inner ear's sensory organs.

 the inner ear is derived from a thickened patch of ectodermal cells, the otic placode, which develops lateral to the developing hindbrain. in birds and mammals, the otic placode invaginates and pinches off to form the pear-shaped otic vesicle, or otocyst. the inner ear's sensory epithelia originate from the ventromedial region of the otocyst, an area that can be defined by the expression of several markers, such as ben, lunatic fringe and cserrate-, islet- and bone morphogentic proteins. the distinctive temporal and spatial expression patterns of bmp in almost all developing chicken inner ear sensory epithelia has led to the suggestion that this protein may play an important role in the induction of inner ear sensory organs. because homozygous bmp knockout mice die between e and e, a period before the inner ear has formed, it is difficult to analyze the role of bmp in the genesis of inner ear sensory organs. as a consequence, other model systems, such as the chicken embryo, have been used to study possible roles of bmp in the inner ear. for example, noggin, an antagonist of bmps, has been employed to interfere with bmp signaling during chicken inner ear development resulting in defects in semicircular canal formation and otic capsule malformation. although malformed or missing cristae were observed in these experiments, the hair cells developed normally. thus these results did not clarify why bmp is robustly expressed in sensory epithelia primordia thereby neither confirming nor refuting the hypothesis that bmp signaling is involved in the genesis of inner ear sensory organs. a possible explanation for the previously observed lack of sensory epithelia defects after in ovo application of bmp antagonists is that the antagonists did not penetrate far enough to reach sufficiently high concentrations in the developing sensory epithelia to block bmp signaling effectively. to address this issue, we exploited a serum-free floating otocyst culture system, which allowed us to quantitatively analyze progenitor cell proliferation, apoptosis and cell differentiation in the developing otocyst with loss of function and gain of function experiments. our experiments revealed that bmp signaling is involved in generation of sensory epithelia by negatively regulating inner ear progenitor cell proliferation through downregulation of the homeodomain transcription factor pax- subsequently, bmp signaling promotes hair cell differentiation.  exogenous bmp leads to increase in hair cell numbers and blockade of bmp signaling inhibits hair cell generation maintenance of the developing avian inner ear in vitro in an environment with largely reduced extrinsic influences can be facilitated by removal of periotic mesenchyme, which otherwise could serve as source for various signaling molecules. as much as possible, we assured removal of the mesenchymal tissue surrounding the otocyst by mild enzymatic treatment with trypsin and careful dissection. we found that removal of the periotic mesenchyme did not affect the capability of stage otic vesicles to generate major inner ear cell types after 7d in serum-free suspension culture. vesicles with obvious mesenchymal contributions that gave rise to cells with chondrocyte morphology were excluded from this study. only otic vesicles that did not display any obvious sign of mesenchymal contribution after culture were included in this study. although free-floating otocysts did not differentiate into morphologically well-defined and structured inner ears, they displayed remarkable constancy with regard to the number of total cells after days in culture and of the number of hair cells formed during the culture period. we identified hair cells with antibody to myosinviia and by their hair bundles labeled with antibody to the hair bundle markers espin and hair cell antigen. furthermore, the hair cells that differentiated in free-floating otocysts appeared to be contacted by neurites, identified with antibody to neuron-specific β-iii tubulin. to assess the effects of bmp signaling on otocyst cells and hair cell generation, we analyzed complete sets of serial sections obtained from individual specimens of each experimental group. when exogenous bmp was added on culture day three, we observed a substantial increase in the number of hair cells, when compared with the untreated controls; hair cells were identified by double-immunostaining for myosinviia and hair cell antigen. blocking of bmp signaling with either noggin or soluble bmp receptor 1a or 1b proteins dramatically reduced hair cell numbers after seven days in culture. the effect of noggin on hair cell generation was dosage-dependant and could be rescued by adding exogenous bmp. exposing cultures to different concentrations of bmp revealed an optimal concentration of 3– ng/ml, which yielded the greatest number of hair cells. bmp inhibits otocyst cell proliferation and induces apoptosis in prospective inner ear ganglion cells to elucidate the effect of bmp on the increased generation of hair cells, we analyzed cell proliferation and apoptosis in cultures treated with exogenous bmp from day three in culture onward. we found that proliferation was significantly reduced in otocysts treated with bmp whereas apoptosis was increased. bmp4-induced apoptosis almost exclusively occurred in areas that we identified as neuronal using the strong islet- immunostaining of cochleovestibular neuron nuclei. islet- is expressed in the developing inner ear in neurons as well as in the nascent sensory epithelia; neuronal islet- staining can easily be distinguished from islet-1-positive developing sensory epithelia by a more round shape of stained neuronal nuclei and higher intensity when compared to epithelial staining. the paired-box transcription factor pax- is expressed in developing sensory epithelia and is down-regulated in cochleovestibular ganglion cells and accordingly, neuronal domains in cultured otocysts lack pax-2-expression. when bmp signaling was blocked with noggin, there was no significant increase in overall cell proliferation, but less apoptosis occurred in the neuronal area, identified by staining for β-iii tubulin. based on these results, we rule out that the increase in hair cell numbers in response to bmp is based on a higher rate of cell proliferation or lower rate of apoptosis. otocyst cells fail to commit to a sensory epithelial fate in the absence of bmp signaling the sensory epithelia of the inner ear, including hair cells and supporting cells, are derived from pax-2-positive progenitor cells that also give rise to other inner ear cell types. the reduction in the number of hair cells in response to blockade of bmp signaling with noggin was not accompanied by a detectable overall reduction of pax-2-positive progenitor cells. as the sensory epithelium progenitor cells become committed to form sensory patches, pax- expression decreases and islet- becomes detectable in both nascent hair cells and supporting cells before the onset of expression of hair cell markers. we found that in noggin-treated otocysts, islet-1-positive epithelial patches containing hair cells appeared noticeably diminished, whereas the more strongly islet-expressing cochleovestibular neurons were apparently not affected. these data support our hypothesis that the generation of sensory patches from pax-2-expressing progenitors depends on bmp signaling. bmp inhibits cell proliferation through downregulation of pax-2 the expression of pax- in the early developing inner ear is usually correlated with increased cell proliferation and absence of apoptosis, which mostly occurs outside of the pax-2-positive regions. when bmp was added at the beginning of the 7d culture period, we noticed a substantial decrease in the number of pax-2-positive cells, although without an apparent effect on the developing sensory epithelium. western blot analysis corroborated that bmp down-regulates pax- expression in a dose-dependent manner. in the normal inner ear, bmp mrna is used as a marker to visualize presumptive sensory epithelium. at the crucial time during development, when sensory epithelia become postmitotic, epithelial islet- expression overlaps with bmp4-expressing areas. conversely, in the same presumptive sensory epithelia, pax- expression is downregulated, suggesting that bmp acts as suppressor of pax- expression during normal inner sensory epithelium formation. discussion bmp signaling is required for inner ear sensory organ formation the discovery of bmp mrna expression in all sensory organ primordia of the developing inner ear has led to the hypothesis that this signaling protein plays a role in the induction or differentiation of inner ear sensory epithelia. however, because homozygous bmp knockout mice do not live beyond the ninth day of embryonic development it is not feasible to analyze bmp function during murine otogenesis. to circumvent this problem, we used a serum-free floating culture technique, which allowed us to quantitatively test the function of bmp on avian inner ear sensory epithelium formation in a controlled environment. this culture regimen enabled us to quantitatively analyze the generation of inner ear cell types by using cell-specific markers. we found that the number of hair cells that formed in otocysts cultured for days is considerably lower than the number of hair cells that can be found in a chicken ear at the 10th day of embryonic development. nevertheless, we argue that the chicken inner ear at e is also substantially larger than the 7-day cultured otocyst, which makes it difficult to assess whether a ratio of ~ % hair cells of the total cell number in a floating otocyst is comparable to the ratio of hair cells versus total inner ear cell number at e nevertheless, we suggest that that the effects we observed in vitro are at least partially representative of the hair cell populations found in the various hair cell-bearing organs of the developing chicken ear. to the best of our ability, we removed periotic mesenchyme from the e otocysts. specimens with obvious mesenchymal contribution were not scored in this study. nevertheless, we did not observe a noticeable difference in the number of hair cells and neurons detectable in otocysts with clear mesenchymal contribution, which is an indication that the periotic mesenchyme appears to exert little influence on the otocyst after stage. loss of bmp signaling by application of noggin or a combination of dominant-negative bmp receptors markedly reduced the number of hair cells in the developing otocyst, which strongly supports the hypothesis that bmp signaling is required for hair cell generation. nevertheless, even at the highest concentrations of noggin or dominant-negative bmp receptors, no complete blockade of hair cell generation was achieved. we suspect that the nature of bmp signaling in the developing sensory epithelia does not allow complete blockade with application of soluble inhibitors. bmp is a secreted protein that binds to cell surface receptors to activate the smad signaling pathway. secreted bmp acts on neighboring cells in a paracrine manner as well as in autocrine fashion on the cells that produce the factor if these cells have appropriate bmp receptors. we hypothesize that extracellular bmp inhibitors cannot completely interfere with autocrine signaling, resulting in an incomplete blockade of bmp signaling in otocyst cells that express both bmp and bmp receptors. interference with bmp signaling in the inner ear in vivo by application of noggin or noggin-producing cells led to inhibition or malformation of semicircular canals accompanied by occasional deformation of ampullae. no effects on sensory epithelium formation and hair cells were reported; however, these studies did not explicitly quantify the number of hair cells. alternatively, we hypothesize that dorso-lateral transplantation of beads soaked with noggin or of noggin-producing cells into the periotic mesenchyme apparently did not suffice to reach sufficient concentrations of the inhibitor over the course of several days in the ventro-medial region of the otocyst, where the sensory epithelia originate. using a serum-free floating culture environment allowed us to control the concentration of bmp or bmp signaling inhibitors more precisely than previous in ovo experiments. bmp promotes hair cell differentiation three mechanisms could account for increase of the number of hair cells by exogenous bmp4: 1) promotion of sensory epithelial progenitor cell proliferation, 2) prevention of progenitor cell apoptosis, or 3) promotion of progenitor cell differentiation to hair cells. our results suggest that bmp inhibits otocyst cell proliferation, but affects apoptosis only in the neuronal domain of cultured otocycts, implying that a plausible mechanism of bmp4's effect on increasing the number of hair cells is to promote sensory epithelium progenitor cells to differentiate. another line of evidence for bmp4's effect on sensory epithelium progenitor cells arises from our loss-of-function analysis with noggin. here we found that noggin-treatment did not affect the number of pax-2-positive cells, whereas the number of hair cells was considerably reduced. this result implies that pax-2-positive progenitors fail to differentiate into sensory epithelium when bmp signaling is blocked. our observations that bmp4-treatment leads to increased hair cell generation and to reduction of otocysts cell proliferation, suggests a dual, concentration-dependant, action of bmp4: at concentrations of 3– ng/ml bmp augments cell differentiation. in fact, we did not find a significant inhibition of cell proliferation with bmp concentrations below ng/ml. at higher bmp concentrations, cell proliferation is sufficiently reduced to offset the bmp effect on hair cell differentiation. the result of the concentration-dependant dual effects of bmp is manifested in a bell-shaped dose-dependency of hair cell numbers. the effects of bmp on otocyst cell proliferation and apoptosis that we report here differ from, but do not contradict, results obtained by in vivo administration of noggin to the developing chicken inner ear. in these experiments, cell proliferation decreased and apoptosis increased after noggin-secreting beads or cells were transplanted into the mesenchyme adjacent to the otocysts. we argue that these previous analyses and our experimental protocol are not easily comparable as many factors could account for the observed differences. for example, interactions of noggin with potential signals in the periotic mesenchyme might produce different effects on inner ear tissue when compared with mesenchyme-free otocyst cultures. another possible explanation is that our analysis included all otocyst-derived cells, whereas previous studies focused on the parts of the developing inner ear surrounding the noggin-secreting grafts. the effect of bmp on promoting differentiation of sensory epithelium progenitor cells is consistent with its temporal and spatial expression in the inner ear sensory organ development. bmp mrna is expressed in all sensory organ primordia before the hair cells and supporting cells are developed. at this crucial period of sensory organ formation, the progenitor cells downregulate pax- leave the cell cycle, and initiate differentiation into hair cells and supporting cells; pax- expression subsequently persists in hair cells. these events appear to be correlated with the presence of bmp in sensory patches, supporting a possible role for bmp in controlling proliferation and specifying differentiation in the inner ear's sensory epithelia. additional clues about the potential targets of bmp in the developing inner ear could arise from analysis of expression of bmp receptors and components of the bmp signaling pathways. beside a comprehensive study in the developing zebrafish ear and lateral line, only one study has addressed in the developing chicken inner ear expression of bmp receptors and smad proteins, the intracellular transducers of bmp signaling. although the latter study was focused on the role of bmp signaling during development of semicircular canals, it should be noted that the expression of bmp receptors in the developing chicken inner ear is probably not restricted to sensory patches or the pax-2-positive domain. a more widespread expression of bmp receptors could potentially be the reason for the substantial downregulation of pax- protein that we observed in free-floating otocycts in response to bmp treatment. at least in the developing zebrafish inner ear several bmp receptors are expressed widely, some of them ubiquitously, at all developmental stages. bmp is a crucial control factor of proliferation, differentiation and apoptosis in the developing inner ear at early stages of inner ear development, all cells of the otic placode and the otic pit express the transcriptional regulator pax- subsequently and noticeably evident at embryonic day three, pax-2-expression gets more localized to the medio-ventral part of the otic vesicle, the region where the sensory epithelia originate. at this time of development, cell proliferation mostly occurs in the pax- expression domain and apoptosis happens outside of the pax- positive domain. we show that the numbers of pax-2-positive cells as well as proliferating cells are markedly reduced when exogenous bmp is added to cultured otocysts. coinciding with decreasing numbers of pax-2-positive cells, we also detected substantial downregulation of the expression of pax- protein. in the developing sensory patches, we were able to visualize that bmp mrna is detectable in the incipient sensory patches just at the time when islet- expression becomes evident. the onset of islet- expression in sensory patches coincides with early differentiation of sensory epithelial cells. in parallel, pax- protein is markedly downregulated in these patches, implying that bmp also causes downregulation of pax- cessation of proliferation, and upregulation of markers for early differentiating sensory epithelium in vivo.  in summary, our results indicate that bmp signaling is involved in the generation of inner ear sensory patches and hair cells. we also found evidence that bmp can affect cell proliferation and apoptosis in different cell populations of the developing inner ear. we therefore conclude that correct control of bmp signaling in all areas of the developing otocyst is central to obtaining an accurate balance of cell proliferation, apoptosis and cell differentiation needed for correct morphogenesis of the inner ear's sensory organs. 
conversely, we observed an increase in the number of hair cells when cultured otocysts were treated with exogenous bmp bmp treatment additionally prompted down-regulation of pax- protein in proliferating sensory epithelial progenitors, leading to reduced progenitor cell proliferation. our results implicate bmp in two events during chicken inner ear sensory epithelium formation: first, in inducing the switch from proliferative sensory epithelium progenitors to differentiating epithelial cells and secondly, in promoting the differentiation of hair cells within the developing sensory epithelia. here we use a chicken otocyst culture system to perform quantitative studies on the development of inner ear cell types and show that hair cell and supporting cell generation is remarkably reduced when bmp signaling is blocked, either with its antagonist noggin or by using soluble bmp receptors. the robust expression of bmp in the incipient sensory organs of the inner ear suggests possible roles for this signaling protein during induction and development of auditory and vestibular sensory epithelia.
 the robust expression of bmp in the incipient sensory organs of the inner ear suggests possible roles for this signaling protein during induction and development of auditory and vestibular sensory epithelia. homozygous bmp4-/- animals die before the inner ear's sensory organs develop, which precludes determining the role of bmp in these organs with simple gene knockout experiments.  here we use a chicken otocyst culture system to perform quantitative studies on the development of inner ear cell types and show that hair cell and supporting cell generation is remarkably reduced when bmp signaling is blocked, either with its antagonist noggin or by using soluble bmp receptors. conversely, we observed an increase in the number of hair cells when cultured otocysts were treated with exogenous bmp bmp treatment additionally prompted down-regulation of pax- protein in proliferating sensory epithelial progenitors, leading to reduced progenitor cell proliferation.  our results implicate bmp in two events during chicken inner ear sensory epithelium formation: first, in inducing the switch from proliferative sensory epithelium progenitors to differentiating epithelial cells and secondly, in promoting the differentiation of hair cells within the developing sensory epithelia. 