BACKGROUND
in the mouse the malleus and incus of the middle ear initially develop as a single element that expresses cartilage markers such as type ii collagen and sox <dig>  this united structure then subdivides to form the two ossicles divided by the incudomalleal joint  <cit> .
this early joint region is free of type ii collagen or sox <dig> expressing cells and expresses joint markers such as gdf <dig>  <cit> .
sox <dig> has been shown to upregulate type ii collagen expression, and overexpression of sox <dig> leads to ectopic cartilage formation  <cit> .
loss of sox <dig> and type ii collagen, therefore, is thought to play an important role in formation of the joint.
the formation of three ossicles in the middle ear  is a characteristic of mammals.
during the course of evolution the primary jaw articulation of non-mammalian vertebrates was replaced by a second articulation between two membranous bones, the squamosal and dentary  <cit> .
studies involving comparative anatomy, embryology and paleontology have suggested that the primary jaw articulation, along with the hyomandibular  were incorporated into the middle ear to form a three ossicle chain.
using this criteria, the malleus is homologous to the articular part of meckel's cartilage, the incus is homologous to the quadrate/palatoquadrate, and the stapes is homologous to the hypomandibular  <cit> .
the incudomalleal joint is therefore homologous to the primary jaw joint.
homology has been confirmed by investigating expression of genes such as bapx <dig> , which is specifically expressed in the primary jaw joint of xenopus, zebrafish and chick and in the incudomalleal joint in mammals  <cit> .
like the incudomalleal joint, the primary jaw joint divides two initially continuous elements expressing type ii collagen, the quadrate and articular part of meckel's cartilage  <cit> .
these two cartilages then separate to form the articulation point for the upper and lower jaws.
in a variety of human syndromes, such as treacher collins and branchio-oto-renal  syndrome, the malleus and incus are often fused resulting in conductive hearing loss  <cit> .
the development of this joint is therefore essential to ensure correct hearing.
it is thus of interest to examine what happens to these presumptive joint cells that are initially fated to become cartilage.
in a variety of limb joints, apoptosis has been observed within the interzone at the centre of the developing joint  <cit> .
such programmed cell death was postulated to account for the loss of the cartilage lineage cells within the forming joint, leading to the separation of skeletal elements  <cit> .
given this data from the limb, we wished to investigate the role of apoptosis in early joint formation in the middle ear.
apoptosis can be mediated by distinct pathways initiating from within  or outside  the cell.
both pathways involve signalling via a family of cysteine proteases named caspases  <cit> .
caspase machinery becomes activated in a cascade manner starting with apical caspases, such as caspase  <dig> and caspase  <dig>  leading to cleavage of effector caspases, such as caspase  <dig>  caspase  <dig> mutants die at e <dig>  due to cardiovascular abnormalities  <cit> , but caspase  <dig> and caspase  <dig> mutant mice survive up to and past birth depending on the strain  <cit> .
these mutants display a strikingly similar phenotype, characterised by a prominent brain malformation caused by defective apoptosis in the proliferative neuroepithelium  <cit> .
caspase  <dig> is activated within a complex known as the apoptosome, where the pro-caspase  <dig> interacts with cytochrome c and apaf- <dig>  <cit> .
cytochrome c deficient mice die at e <dig>   <cit> , however the majority of apaf- <dig> mutant mice survive up to e <dig> .
these mice exhibit defects in the brain, inner ear and digits  <cit> .
the middle ear can therefore be analysed in apaf- <dig>  caspase  <dig>  and caspase  <dig> mutant mice.
to knock down activation of all caspases, an in vitro approach must be applied and pharmacological inhibitors binding to the active site of activated caspases are generally used  <cit> .
we have therefore devised a method for culturing the middle ear in the presence or absence of caspase activity.
by these two methods the role of apoptosis in formation of the incudomalleal joint can be analysed.
in the digits, developing cartilage recruits neighbouring cells into the cartilage creating high cell density in these regions  <cit> .
it was therefore possible that the sox9/type ii collagen expressing cells in the presumptive joint might migrate out of the forming joint into the body of the malleus or incus.
to test this we labelled cells in the forming incudomalleal joint region and developing malleus with the lipophilic dyes dio and dii, and observed the movements of labelled cells as the joint formed in culture.
alternatively, the sox9/type ii collagen positive cells might remain in position and downregulate expression of these genes.
to investigate this possibility we have utilised type ii collagen lacz reporter mice, where the stable lacz protein marks cells that express type ii collagen and also those that have recently switched off type ii collagen.
by these methods we hope to determine what processes are involved in early joint development in the middle ear.
methods
animals
wild type and type ii collagen lacz reporter mouse embryos were dissected out at embryonic day   <dig>  to  <dig> .
caspase 3/c57bi/6j mutant mice were provided by dr. s. lakhani .
caspase 9/c57bi/6j mutant mice were provide by prof. k.a.
roth .
apaf- <dig> mutant mice were generated as described in  <cit> .
all research on animals follows internationally recognized guidelines.
use of genetically modified organisms was approved by the local genetic modification safety committee .
middle ear explant culture system
middle ear regions were dissected from the lateral parts of mouse embryonic heads, and placed on millipore filters supported on metal grids over medium using a modified trowell method  <cit> .
culture medium consisted of dmem  supplemented with glutamine, pen/strep and 10% fetal calf serum.
fresh medium was delivered every  <dig> h and cultures were kept at 37°c, 5% co <dig> for up to  <dig> days.
caspase inhibition
the pharmaceutical inhibitor of general caspase z-vad-fmk  was dissolved in acs dmso  and added to the fresh culture medium at a final concentration of  <dig> μm.
control medium was supplemented by 1%dmso corresponding to the amount of dmso in the inhibitor solution, as has been used in previous publications  <cit> .
culture medium was changed every day for  <dig> days.
biotinylated pan caspase inhibitor  was exploited to check the distribution of the inhibitor throughout the explant.
the pan caspase inhibitor was applied in the same way as the general one and its distribution was traced in histological sections after addition of streptavidin-ap  and nbt/bcip colour reaction, showing successful spread of the inhibitor .
the effect of the inhibitor on apoptosis was tested in developing limbs where the interdigital tissue is known to disappear by caspase dependent apoptosis  <cit> .
front limbs from e <dig>  mouse embryos were cultured simultaneously and in the same way as the middle ear cultures for 24– <dig> hours.
formation of digits and interdigital space was documented microscopically.
staining
embryos were fixed in 4% paraformaldehyde and dehydrated to 100% ethanol.
paraffin wax sections were cut at  <dig> μm and split over  <dig> to  <dig> slides.
serial sections were stained with hematoxylin/eosin to visualize the early tissue morphology or with sirrus red and alcian blue to view the developing bones and cartilage.
type ii collagen lacz reporter mice were fixed and stained with β galactosidase in wholemount as described in  <cit> .
proliferation assays
pcna
a polyclonal pcna antibody  was applied for  <dig> hour  after pretreatment in  <dig>  m citric buffer .
application of the secondary antibody  for  <dig> mins was followed by a colour reaction using 3', <dig> '-diaminobensidine  as the substrate.
anti-phosphohistone h <dig> immunohistochemistry
embryos were fixed in 5% acetic acid in ethanol overnight at 4°c, dehydrated through a graded series of ethanol, embedded in paraffin wax and sectioned.
sections were processed for antibody staining using rabbit anti-phosphorylated histone  <dig>  as described previously  <cit> .
the antibody targets were visualized with alexa-fluor 568-goat-anti-rabbit-igg .
slides were mounted in slowfade gold antifade reagent with dapi  according to manufacturers instructions and visualized by fluorescence microscopy .
apoptosis evaluation
tunel assay
an apoptag peroxidase in situ apoptosis detection kit was used to detect apoptotic dna breaks in individual cells .
for the colour reaction a dab substrate kit was used, with nickel staining to give a black colour .
eosin was used for counterstaining of the sections.
morphological criteria
nuclear condensations and formation of apoptotic bodies were evaluated additionally to the tunel test  <cit> .
cell labelling
dii  and dio  were used as cell lineage labels.
a stock solution of dii   was made in 100% ethanol and diluted 1: <dig> with  <dig>  m sucrose on the day of use.
for the dio labelling, a ready-made vybrant dio cell-labelling solution was used .
the dil solution was injected into the developing malleus of dissected lower jaws using a mouth pipette connected to a pulled glass needle.
by a similar method dio was injected into the presumptive joint region between the malleus and incus.
the cultures were then moved to explant culture dishes and photographed under light and dark field.
cultures were left to develop for  <dig> hours and photographed at  <dig> hour intervals.
type ii collagen immunohistochemistry
type ii collagen immunohistochemistry was performed using 11–116b <dig> antibody  on paraffin wax sections.
to enhance the signal, slides were microwaved in  <dig>  m citrate buffer  <cit> , and treated with chondroitinase abc at  <dig>  u per ml and hyaluronidase at  <dig>  u per ml at 37°c for  <dig> minutes .
the collagen antibody was used at a dilution of 1: <dig>
radioactive in situ hybridisation
radioactive in situs were performed using 35s as described  <cit> .
gdf <dig> probe was linearised with hindiii and transcribed with t <dig>
RESULTS
proliferation in the incudomalleal joint
very few pcna positive cells have been observed in the immediate layer of developing joints in the limb  <cit> .
the joint region is thus thought of as a region of low proliferation.
to see whether this situation is mimicked in the middle ear we looked at the distribution of proliferating cells during early formation of the incudomalleal joint.
at e <dig>  and e <dig>  the malleus and incus are seen as separate structures with a clear joint in between.
using pcna, the level of proliferation was high in the head of the malleus and incus but the joint region was negative .
pcna has a long half-life leading to its continuous expression in cells that are not actively dividing  <cit> .
we, therefore, supplemented our results by using anti-phosphohistone h <dig> immunohistochemistry.
the h <dig> antibody detects phosphorylation of histone h <dig>  which is high during chromosome condensation as the cell enters the m-phase and has a short half-life.
as expected, only a few cells were observed as positive in the middle ear using this method compared to using pcna.
the positive cells were concentrated in and around the ossicles but again excluded from the developing joint .
labelled cells were also located around the circumference of the eye as previously published using this method, indicating that the assay was working  <cit> .
at e <dig>  the malleus and incus are still continuous  <cit> .
at this early stage using pcna, proliferation was observed in the malleus and incus but the forming joint region was negative, reflecting the situation observed at later stages .
using the h <dig> antibody at this stage, the incus showed positive proliferating cells but the presumptive joint region appeared negative .
the malleus also appeared negative, using the antibody to h <dig>  perhaps indicating a time delay between proliferation of the two ossicles.
apoptosis in the incudomalleal joint
a tunel assay was performed to detect nuclear dna fragmentation in the developing joint from e <dig>  to e <dig> .
additionally, morphological criteria of apoptotic cells  were evaluated.
at e <dig> , as the malleus and incus are starting to separate, a clear region of apoptotic cells was visible in a whirl underneath the head of the malleus  .
at e <dig>  and e <dig> , when the joint is clear histologically, a few positive cells were located in this same region under the head of the developing malleus, with a line of positive cells running between the malleus and the incus  .
tunel positive cells were also observed in the external auditory meatus and in the inner ear, where apoptosis has been previously described, confirming that the method was successful  <cit>  .
to investigate the functional significance of these areas of apoptosis for middle ear development, we investigated middle ear development in mice where different components of the apoptotic machinery had been knocked out, and used an explant culture system to block the action of all caspases in this region.
pro-apoptotic molecules apaf- <dig>  caspase  <dig> and  <dig> are not involved in the initiation of the incudomalleal joint
mutant mice are a great tool to study the consequences of a particular apoptotic deficiency in vivo.
apaf- <dig>  caspase  <dig> and caspase  <dig> mutants were analysed at e <dig>  to reveal any consequences of their deficiency on early development of the middle ear joint.
these embryos displayed a variety of craniofacial defects, with a number showing obvious overgrowth of the brain, as would be expected from previously published observations  <cit> .
by e <dig>  the joint region between the malleus and incus is clearly visible in section in wildtype mice .
the malleus and incus were found completely separated, and no cartilage-like cells were evident in the joint in either the wildtype or mutant embryos  .
in order to confirm the absence of apoptosis in these mutants a tunel stain was carried out in the apaf- <dig> mutants.
no tunel positive cells were observed in the middle ear region, compared to a heterozygous littermate or wildtype, thus confirming that apoptosis has been inhibited in these mutants .
to confirm the lack of phenotype in the mutants, sections were carried out on apaf- <dig> mutants at e <dig>  and e <dig> , once the shape of the articulation is well formed .
in section the incudomalleal joint appeared normal, but the shape of the articulation showed subtle variations in some specimens while in others it appeared identical to that of a wildtype littermate .
this difference might relate to slight variations in the angle of section, rather than indicate a true defect, and so to confirm the result skeletal preparations of mutant heads were examined.
in these no obvious defect was observed in the shape of the articulation and the body of the malleus and incus appeared normal  .
loss of apoptosis, therefore, did not appear to disrupt joint formation or gross joint morphology.
to confirm this an in vitro explant culture approach was introduced to inhibit all caspases in cultures of the middle ear.
caspase inhibition in middle ear explant culture
pharmaceutical inhibitors bind to the active site of activated caspases and are routinely used to inhibit caspase activity in vitro  <cit> .
for our explant culture we used a modified method of ex vivo middle ear culture  <cit> .
this technique allows a clear observation of the middle ear structures as they develop over several days.
in order to demonstrate the effectiveness of apoptosis inhibition using the general caspase inhibitor, developing front limbs at e <dig>  and e <dig>  were cultured ex vivo for  <dig> hours.
interdigital tissue regression was prevented in the caspase inhibitor samples compared to the controls  .
middle ears were dissected out at e <dig> , several days prior to any morphological sign of a joint.
these were then cultured for  <dig> days until a clear joint could be observed between the malleus and incus in control cultures.
in cultures treated with the general caspase inhibitor a normal joint was observed, free from cartilaginous cells  .
cell migration
utilising the same middle ear explant culture system, we investigated whether cells in the presumptive joint region migrated away from the developing interzone and were recruited into the ossicles.
cultures were set up at e <dig> , before joint initiation.
the ossicles and associated middle ear structures developed well in culture along with neighbouring structures such as the inner ear .
double labelling was used to identify any movement between cells in the presumptive joint region and the neighbouring malleus.
a single spot of dio was mouth pipetted in the region of the presumptive joint and a second spot of dii was placed alongside in the developing malleus  .
the movement of these two groups of labelled cells was followed for  <dig> hours.
over this time period no movement of the dio labelled cells towards the dii labelled cells was observed .
as the presumptive joint region is not clearly defined morphologically, in some cases, a few cells in the presumptive incus were also labelled at the same time as the joint.
in these cases labelled cells could be seen incorporated into the developing incus .
although cells did not appear to migrate from the presumptive joint into the malleus, the joint region itself did change shape and elongated caudally as the ossicles developed.
this does not necessarily represent active migration but a re-shaping of the joint region as it is squeezed by the proliferating ossicles on either side.
downregulation of cartilage markers
from the lack of apoptosis or migration out of the forming joint region it would appear that cells in the joint are turning off a cartilage pathway and turning on a joint specific pathway.
to test this we used type ii collagen reporter mice  to take advantage of the fact that the lacz protein is very stable.
at e <dig>  type ii collagen expression, as assessed by in situ hybridisation or immunohistochemistry, is not observed in the joint region .
instead joint markers, such as gdf <dig>  are expressed at high levels throughout the joint   <cit> .
in contrast, blue cells are still found at high levels in between the two ossicles when the type ii collagen lacz reporter mouse is stained for lacz at this stage .
by combining a lacz stain  and type ii collagen immunohistochemistry , cells that still express type ii collagen will be visualised as both brown and blue, while those cells that have recently turned off type ii collagen will be blue only.
we can thus view the expression history of cells in the joint region.
cells in the joint region at e <dig>  expressed lacz , while the surrounding ossicles expressed both lacz  and the type ii collagen protein  .
thus the type ii collagen expressing cells have remained in the same position and downregulated expression of chondrogenic genes.
discussion
apoptosis has been described as a sculptor of development in many mammalian structures and is observed in developing limb joints  <cit> .
thus it seems a good candidate process for removal of sox9/type ii collagen expressing cells to create a joint, and thereby separate the malleus and incus.
apoptotic cells were observed early on between the malleus and incus, however, apaf- <dig>  caspase  <dig> and caspase  <dig> mutant mice exhibited normal division of the malleus and incus at e <dig> .
no middle ear defect was also observed in apaf- <dig> mutants at e <dig> .
this is despite the loss of tunel positive cells in the mutant.
this result was confirmed in explant culture by using caspase inhibitors.
thus formation of the middle ear joint appears independent of apoptosis.
it may be, however, that loss of apoptosis is compensated for by cell death through other processes, such as necrosis or caspase independent programmed cell death, such as paraptosis  <cit> .
paraptosis, like apoptosis, is programmed cell death but is independent of the normal apoptotic machinery  <cit> .
in the limbs of apaf- <dig> mutants interdigital cell death is initially inhibited but after a delay of a few days, cell death in these regions is observed, resulting in separation of the digits.
this interdigital cell death occurs despite no evidence of tunel positive cells or overall cell condensation  <cit> .
thus in the absence of apoptosis, as indicated by tunel staining, cells may still be able to die.
migration is another candidate process that could explain the disappearance of cartilaginous presumptive joint cells.
dii and dio double labelling was used to trace the movement of cells in the presumptive joint region over  <dig> days.
results show that although the joint region changes shape, the labelled cells in the joint region did not move towards the dii labelled cells and were not incorporated into the malleus.
this is perhaps unsurprising as chondrogenic precursors have been shown to have a low migration capacity when compared to many other cells types, such as myogenic precursors  <cit> .
by comparing the expression of type ii collagen protein and a lacz reporter driven by the same promoter, we have shown that the initially chondrogenic cells remain in the incudomalleal joint region and downregulate expression of chondrogenic markers.
in this way type ii collagen is turned off and joint markers, such as gdf <dig>  are turned on in the same cells.
the signal to trigger this event is as yet unknown.
it is thus unclear how cells only in the presumptive joint region turn off type ii collagen, while those on either side carry on with a cartilage differentiation pathway.
the homeobox gene emx <dig> may play a role in demarcating the future joint as it is expressed in the incus and presumptive joint but excluded from the malleus, prior to the separation of these two ossicles  <cit> .
in keeping with this role, mutations in emx <dig> lead to loss of both the incus and joint  <cit> .
joint markers, such as bapx <dig> and gdf <dig>  have been shown to turn on before loss of sox <dig> and type ii collagen  <cit> .
it is possible, therefore, that these joint markers act by directly repressing cartilage development.
overexpression of gdf <dig>  however, is unable to induce formation of an ectopic joint, thus although this gene has an important role in joint formation it is not sufficient to repress cartilage development  <cit> .
in contrast, overexpression of wnt signalling is sufficient to induce an ectopic joint  <cit> .
the role of wnt signalling in formation of the incudomalleal joint and its relationship to the loss of cartilage markers is thus an interesting avenue to pursue.
what leads to the induction of joint markers at the future incudo-malleal joint is also unclear.
in the chick and zebrafish, bapx <dig> acts upstream of gdf <dig> in development of the primary jaw joint, with the expression of bapx <dig> being controlled by a combination of inhibitory and inducing signals from fgfs, bmps and endothelin  <cit> .
whether a similar combination of signals leads to the restriction of joint markers in the mammalian middle ear is therefore another important area for future study.
such comparative work will allow for further understanding of how cranial joints are initiated and positioned and will focus in on the genetic pathways that may have played a role in changing the pattern of joints during evolution.
CONCLUSIONS
the incudomalleal joint in the mammalian middle ear separates the initially united incus and malleus.
the forming joint is characterised by a lack of proliferation, compared to the ossicles on either side, and contains a number of cells undergoing cell death .
in the absence of apoptosis, however, no defect in joint formation is observed.
cells in the joint region do not move away from this position but instead downregulate cartilage markers and take on a joint fate, expressing genes such as gdf <dig>  fusions of the middle ear, as seen in patients with bor syndrome or treacher collins syndrome, may therefore occur if presumptive joint cells fail to downregulate expression of chondrogenic genes.
many of the genes underlying such syndromes have been discovered, and so the next challenge is to investigate such possibilities using mouse models.
competing interests
the author declare that they have no competing interests.
authors' contributions
sa and ast wrote the manuscript.
sa performed the majority of the experiments described with the help and assistance of at, in whose lab the research was carried out.
em performed the pcna results, provided the caspase mutant sections and wrote the text relating to apoptosis.
cs sectioned apaf- <dig> mutants and performed the anti-phosphohistone h <dig> immunohistochemistry.
hy provided a range of apaf- <dig> mutant embryos.
all authors have read and approved the final manuscript.

 in the mouse the malleus and incus of the middle ear initially develop as a single element that expresses cartilage markers such as type ii collagen and sox this united structure then subdivides to form the two ossicles divided by the incudomalleal joint. this early joint region is free of type ii collagen or sox expressing cells and expresses joint markers such as gdf. sox has been shown to upregulate type ii collagen expression, and overexpression of sox leads to ectopic cartilage formation. loss of sox and type ii collagen, therefore, is thought to play an important role in formation of the joint. the formation of three ossicles in the middle ear is a characteristic of mammals. during the course of evolution the primary jaw articulation of non-mammalian vertebrates was replaced by a second articulation between two membranous bones, the squamosal and dentary. studies involving comparative anatomy, embryology and paleontology have suggested that the primary jaw articulation, along with the hyomandibular were incorporated into the middle ear to form a three ossicle chain. using this criteria, the malleus is homologous to the articular part of meckel's cartilage, the incus is homologous to the quadrate/palatoquadrate, and the stapes is homologous to the hypomandibular. the incudomalleal joint is therefore homologous to the primary jaw joint. homology has been confirmed by investigating expression of genes such as bapx, which is specifically expressed in the primary jaw joint of xenopus, zebrafish and chick and in the incudomalleal joint in mammals. like the incudomalleal joint, the primary jaw joint divides two initially continuous elements expressing type ii collagen, the quadrate and articular part of meckel's cartilage. these two cartilages then separate to form the articulation point for the upper and lower jaws. in a variety of human syndromes, such as treacher collins and branchio-oto-renal syndrome, the malleus and incus are often fused resulting in conductive hearing loss. the development of this joint is therefore essential to ensure correct hearing. it is thus of interest to examine what happens to these presumptive joint cells that are initially fated to become cartilage. in a variety of limb joints, apoptosis has been observed within the interzone at the centre of the developing joint. such programmed cell death was postulated to account for the loss of the cartilage lineage cells within the forming joint, leading to the separation of skeletal elements. given this data from the limb, we wished to investigate the role of apoptosis in early joint formation in the middle ear. apoptosis can be mediated by distinct pathways initiating from within or outside the cell. both pathways involve signalling via a family of cysteine proteases named caspases. caspase machinery becomes activated in a cascade manner starting with apical caspases, such as caspase and caspase leading to cleavage of effector caspases, such as caspase caspase mutants die at e due to cardiovascular abnormalities, but caspase and caspase mutant mice survive up to and past birth depending on the strain. these mutants display a strikingly similar phenotype, characterised by a prominent brain malformation caused by defective apoptosis in the proliferative neuroepithelium. caspase is activated within a complex known as the apoptosome, where the pro-caspase interacts with cytochrome c and apaf-. cytochrome c deficient mice die at e, however the majority of apaf- mutant mice survive up to e. these mice exhibit defects in the brain, inner ear and digits. the middle ear can therefore be analysed in apaf- caspase and caspase mutant mice. to knock down activation of all caspases, an in vitro approach must be applied and pharmacological inhibitors binding to the active site of activated caspases are generally used. we have therefore devised a method for culturing the middle ear in the presence or absence of caspase activity. by these two methods the role of apoptosis in formation of the incudomalleal joint can be analysed. in the digits, developing cartilage recruits neighbouring cells into the cartilage creating high cell density in these regions. it was therefore possible that the sox9/type ii collagen expressing cells in the presumptive joint might migrate out of the forming joint into the body of the malleus or incus. to test this we labelled cells in the forming incudomalleal joint region and developing malleus with the lipophilic dyes dio and dii, and observed the movements of labelled cells as the joint formed in culture. alternatively, the sox9/type ii collagen positive cells might remain in position and downregulate expression of these genes. to investigate this possibility we have utilised type ii collagen lacz reporter mice, where the stable lacz protein marks cells that express type ii collagen and also those that have recently switched off type ii collagen. by these methods we hope to determine what processes are involved in early joint development in the middle ear. methods animals wild type and type ii collagen lacz reporter mouse embryos were dissected out at embryonic day to. caspase 3/c57bi/6j mutant mice were provided by dr. s. lakhani. caspase 9/c57bi/6j mutant mice were provide by prof. k.a. roth. apaf- mutant mice were generated as described in. all research on animals follows internationally recognized guidelines. use of genetically modified organisms was approved by the local genetic modification safety committee. middle ear explant culture system middle ear regions were dissected from the lateral parts of mouse embryonic heads, and placed on millipore filters supported on metal grids over medium using a modified trowell method. culture medium consisted of dmem supplemented with glutamine, pen/strep and 10% fetal calf serum. fresh medium was delivered every h and cultures were kept at 37°c, 5% co for up to days. caspase inhibition the pharmaceutical inhibitor of general caspase z-vad-fmk was dissolved in acs dmso and added to the fresh culture medium at a final concentration of μm. control medium was supplemented by 1%dmso corresponding to the amount of dmso in the inhibitor solution, as has been used in previous publications. culture medium was changed every day for days. biotinylated pan caspase inhibitor was exploited to check the distribution of the inhibitor throughout the explant. the pan caspase inhibitor was applied in the same way as the general one and its distribution was traced in histological sections after addition of streptavidin-ap and nbt/bcip colour reaction, showing successful spread of the inhibitor. the effect of the inhibitor on apoptosis was tested in developing limbs where the interdigital tissue is known to disappear by caspase dependent apoptosis. front limbs from e mouse embryos were cultured simultaneously and in the same way as the middle ear cultures for 24– hours. formation of digits and interdigital space was documented microscopically. staining embryos were fixed in 4% paraformaldehyde and dehydrated to 100% ethanol. paraffin wax sections were cut at μm and split over to slides. serial sections were stained with hematoxylin/eosin to visualize the early tissue morphology or with sirrus red and alcian blue to view the developing bones and cartilage. type ii collagen lacz reporter mice were fixed and stained with β galactosidase in wholemount as described in. proliferation assays pcna a polyclonal pcna antibody was applied for hour after pretreatment in m citric buffer. application of the secondary antibody for mins was followed by a colour reaction using 3','-diaminobensidine as the substrate. anti-phosphohistone h immunohistochemistry embryos were fixed in 5% acetic acid in ethanol overnight at 4°c, dehydrated through a graded series of ethanol, embedded in paraffin wax and sectioned. sections were processed for antibody staining using rabbit anti-phosphorylated histone as described previously. the antibody targets were visualized with alexa-fluor 568-goat-anti-rabbit-igg. slides were mounted in slowfade gold antifade reagent with dapi according to manufacturers instructions and visualized by fluorescence microscopy. apoptosis evaluation tunel assay an apoptag peroxidase in situ apoptosis detection kit was used to detect apoptotic dna breaks in individual cells. for the colour reaction a dab substrate kit was used, with nickel staining to give a black colour. eosin was used for counterstaining of the sections. morphological criteria nuclear condensations and formation of apoptotic bodies were evaluated additionally to the tunel test. cell labelling dii and dio were used as cell lineage labels. a stock solution of dii was made in 100% ethanol and diluted 1: with m sucrose on the day of use. for the dio labelling, a ready-made vybrant dio cell-labelling solution was used. the dil solution was injected into the developing malleus of dissected lower jaws using a mouth pipette connected to a pulled glass needle. by a similar method dio was injected into the presumptive joint region between the malleus and incus. the cultures were then moved to explant culture dishes and photographed under light and dark field. cultures were left to develop for hours and photographed at hour intervals. type ii collagen immunohistochemistry type ii collagen immunohistochemistry was performed using 11–116b antibody on paraffin wax sections. to enhance the signal, slides were microwaved in m citrate buffer, and treated with chondroitinase abc at u per ml and hyaluronidase at u per ml at 37°c for minutes. the collagen antibody was used at a dilution of 1: radioactive in situ hybridisation radioactive in situs were performed using 35s as described. gdf probe was linearised with hindiii and transcribed with t  proliferation in the incudomalleal joint very few pcna positive cells have been observed in the immediate layer of developing joints in the limb. the joint region is thus thought of as a region of low proliferation. to see whether this situation is mimicked in the middle ear we looked at the distribution of proliferating cells during early formation of the incudomalleal joint. at e and e the malleus and incus are seen as separate structures with a clear joint in between. using pcna, the level of proliferation was high in the head of the malleus and incus but the joint region was negative. pcna has a long half-life leading to its continuous expression in cells that are not actively dividing. we, therefore, supplemented our results by using anti-phosphohistone h immunohistochemistry. the h antibody detects phosphorylation of histone h which is high during chromosome condensation as the cell enters the m-phase and has a short half-life. as expected, only a few cells were observed as positive in the middle ear using this method compared to using pcna. the positive cells were concentrated in and around the ossicles but again excluded from the developing joint. labelled cells were also located around the circumference of the eye as previously published using this method, indicating that the assay was working. at e the malleus and incus are still continuous. at this early stage using pcna, proliferation was observed in the malleus and incus but the forming joint region was negative, reflecting the situation observed at later stages. using the h antibody at this stage, the incus showed positive proliferating cells but the presumptive joint region appeared negative. the malleus also appeared negative, using the antibody to h perhaps indicating a time delay between proliferation of the two ossicles. apoptosis in the incudomalleal joint a tunel assay was performed to detect nuclear dna fragmentation in the developing joint from e to e. additionally, morphological criteria of apoptotic cells were evaluated. at e, as the malleus and incus are starting to separate, a clear region of apoptotic cells was visible in a whirl underneath the head of the malleus. at e and e, when the joint is clear histologically, a few positive cells were located in this same region under the head of the developing malleus, with a line of positive cells running between the malleus and the incus. tunel positive cells were also observed in the external auditory meatus and in the inner ear, where apoptosis has been previously described, confirming that the method was successful. to investigate the functional significance of these areas of apoptosis for middle ear development, we investigated middle ear development in mice where different components of the apoptotic machinery had been knocked out, and used an explant culture system to block the action of all caspases in this region. pro-apoptotic molecules apaf- caspase and are not involved in the initiation of the incudomalleal joint mutant mice are a great tool to study the consequences of a particular apoptotic deficiency in vivo. apaf- caspase and caspase mutants were analysed at e to reveal any consequences of their deficiency on early development of the middle ear joint. these embryos displayed a variety of craniofacial defects, with a number showing obvious overgrowth of the brain, as would be expected from previously published observations. by e the joint region between the malleus and incus is clearly visible in section in wildtype mice. the malleus and incus were found completely separated, and no cartilage-like cells were evident in the joint in either the wildtype or mutant embryos. in order to confirm the absence of apoptosis in these mutants a tunel stain was carried out in the apaf- mutants. no tunel positive cells were observed in the middle ear region, compared to a heterozygous littermate or wildtype, thus confirming that apoptosis has been inhibited in these mutants. to confirm the lack of phenotype in the mutants, sections were carried out on apaf- mutants at e and e, once the shape of the articulation is well formed. in section the incudomalleal joint appeared normal, but the shape of the articulation showed subtle variations in some specimens while in others it appeared identical to that of a wildtype littermate. this difference might relate to slight variations in the angle of section, rather than indicate a true defect, and so to confirm the result skeletal preparations of mutant heads were examined. in these no obvious defect was observed in the shape of the articulation and the body of the malleus and incus appeared normal. loss of apoptosis, therefore, did not appear to disrupt joint formation or gross joint morphology. to confirm this an in vitro explant culture approach was introduced to inhibit all caspases in cultures of the middle ear. caspase inhibition in middle ear explant culture pharmaceutical inhibitors bind to the active site of activated caspases and are routinely used to inhibit caspase activity in vitro. for our explant culture we used a modified method of ex vivo middle ear culture. this technique allows a clear observation of the middle ear structures as they develop over several days. in order to demonstrate the effectiveness of apoptosis inhibition using the general caspase inhibitor, developing front limbs at e and e were cultured ex vivo for hours. interdigital tissue regression was prevented in the caspase inhibitor samples compared to the controls. middle ears were dissected out at e, several days prior to any morphological sign of a joint. these were then cultured for days until a clear joint could be observed between the malleus and incus in control cultures. in cultures treated with the general caspase inhibitor a normal joint was observed, free from cartilaginous cells. cell migration utilising the same middle ear explant culture system, we investigated whether cells in the presumptive joint region migrated away from the developing interzone and were recruited into the ossicles. cultures were set up at e, before joint initiation. the ossicles and associated middle ear structures developed well in culture along with neighbouring structures such as the inner ear. double labelling was used to identify any movement between cells in the presumptive joint region and the neighbouring malleus. a single spot of dio was mouth pipetted in the region of the presumptive joint and a second spot of dii was placed alongside in the developing malleus. the movement of these two groups of labelled cells was followed for hours. over this time period no movement of the dio labelled cells towards the dii labelled cells was observed. as the presumptive joint region is not clearly defined morphologically, in some cases, a few cells in the presumptive incus were also labelled at the same time as the joint. in these cases labelled cells could be seen incorporated into the developing incus. although cells did not appear to migrate from the presumptive joint into the malleus, the joint region itself did change shape and elongated caudally as the ossicles developed. this does not necessarily represent active migration but a re-shaping of the joint region as it is squeezed by the proliferating ossicles on either side. downregulation of cartilage markers from the lack of apoptosis or migration out of the forming joint region it would appear that cells in the joint are turning off a cartilage pathway and turning on a joint specific pathway. to test this we used type ii collagen reporter mice to take advantage of the fact that the lacz protein is very stable. at e type ii collagen expression, as assessed by in situ hybridisation or immunohistochemistry, is not observed in the joint region. instead joint markers, such as gdf are expressed at high levels throughout the joint. in contrast, blue cells are still found at high levels in between the two ossicles when the type ii collagen lacz reporter mouse is stained for lacz at this stage. by combining a lacz stain and type ii collagen immunohistochemistry, cells that still express type ii collagen will be visualised as both brown and blue, while those cells that have recently turned off type ii collagen will be blue only. we can thus view the expression history of cells in the joint region. cells in the joint region at e expressed lacz, while the surrounding ossicles expressed both lacz and the type ii collagen protein. thus the type ii collagen expressing cells have remained in the same position and downregulated expression of chondrogenic genes. discussion apoptosis has been described as a sculptor of development in many mammalian structures and is observed in developing limb joints. thus it seems a good candidate process for removal of sox9/type ii collagen expressing cells to create a joint, and thereby separate the malleus and incus. apoptotic cells were observed early on between the malleus and incus, however, apaf- caspase and caspase mutant mice exhibited normal division of the malleus and incus at e. no middle ear defect was also observed in apaf- mutants at e. this is despite the loss of tunel positive cells in the mutant. this result was confirmed in explant culture by using caspase inhibitors. thus formation of the middle ear joint appears independent of apoptosis. it may be, however, that loss of apoptosis is compensated for by cell death through other processes, such as necrosis or caspase independent programmed cell death, such as paraptosis. paraptosis, like apoptosis, is programmed cell death but is independent of the normal apoptotic machinery. in the limbs of apaf- mutants interdigital cell death is initially inhibited but after a delay of a few days, cell death in these regions is observed, resulting in separation of the digits. this interdigital cell death occurs despite no evidence of tunel positive cells or overall cell condensation. thus in the absence of apoptosis, as indicated by tunel staining, cells may still be able to die. migration is another candidate process that could explain the disappearance of cartilaginous presumptive joint cells. dii and dio double labelling was used to trace the movement of cells in the presumptive joint region over days. results show that although the joint region changes shape, the labelled cells in the joint region did not move towards the dii labelled cells and were not incorporated into the malleus. this is perhaps unsurprising as chondrogenic precursors have been shown to have a low migration capacity when compared to many other cells types, such as myogenic precursors. by comparing the expression of type ii collagen protein and a lacz reporter driven by the same promoter, we have shown that the initially chondrogenic cells remain in the incudomalleal joint region and downregulate expression of chondrogenic markers. in this way type ii collagen is turned off and joint markers, such as gdf are turned on in the same cells. the signal to trigger this event is as yet unknown. it is thus unclear how cells only in the presumptive joint region turn off type ii collagen, while those on either side carry on with a cartilage differentiation pathway. the homeobox gene emx may play a role in demarcating the future joint as it is expressed in the incus and presumptive joint but excluded from the malleus, prior to the separation of these two ossicles. in keeping with this role, mutations in emx lead to loss of both the incus and joint. joint markers, such as bapx and gdf have been shown to turn on before loss of sox and type ii collagen. it is possible, therefore, that these joint markers act by directly repressing cartilage development. overexpression of gdf however, is unable to induce formation of an ectopic joint, thus although this gene has an important role in joint formation it is not sufficient to repress cartilage development. in contrast, overexpression of wnt signalling is sufficient to induce an ectopic joint. the role of wnt signalling in formation of the incudomalleal joint and its relationship to the loss of cartilage markers is thus an interesting avenue to pursue. what leads to the induction of joint markers at the future incudo-malleal joint is also unclear. in the chick and zebrafish, bapx acts upstream of gdf in development of the primary jaw joint, with the expression of bapx being controlled by a combination of inhibitory and inducing signals from fgfs, bmps and endothelin. whether a similar combination of signals leads to the restriction of joint markers in the mammalian middle ear is therefore another important area for future study. such comparative work will allow for further understanding of how cranial joints are initiated and positioned and will focus in on the genetic pathways that may have played a role in changing the pattern of joints during evolution.  the incudomalleal joint in the mammalian middle ear separates the initially united incus and malleus. the forming joint is characterised by a lack of proliferation, compared to the ossicles on either side, and contains a number of cells undergoing cell death. in the absence of apoptosis, however, no defect in joint formation is observed. cells in the joint region do not move away from this position but instead downregulate cartilage markers and take on a joint fate, expressing genes such as gdf fusions of the middle ear, as seen in patients with bor syndrome or treacher collins syndrome, may therefore occur if presumptive joint cells fail to downregulate expression of chondrogenic genes. many of the genes underlying such syndromes have been discovered, and so the next challenge is to investigate such possibilities using mouse models. competing interests the author declare that they have no competing interests. authors' contributions sa and ast wrote the manuscript. sa performed the majority of the experiments described with the help and assistance of at, in whose lab the research was carried out. em performed the pcna results, provided the caspase mutant sections and wrote the text relating to apoptosis. cs sectioned apaf- mutants and performed the anti-phosphohistone h immunohistochemistry. hy provided a range of apaf- mutant embryos. all authors have read and approved the final manuscript. 
the incudomalleal joint region forms by cells in the presumptive joint region switching off cartilage markers and turning on joint markers. apoptosis has been suggested to aid in the removal of pre-cartilaginous cells from the joint region, allowing for the physical separation of the cartilaginous elements, however, we show that joint initiation is unaffected by blocking apoptosis. there is also no evidence of cell migration out of the presumptive joint region, as observed by labelling of joint and ossicle cells in culture. using type ii collagen lacz reporter mice, however, it is evident that cells in the presumptive joint region remain in place and downregulate cartilage markers. the middle ear of mammals is composed of three endochondrial ossicles, the stapes, incus and malleus. fusion of the malleus and incus is observed in a number of human syndromes and results in conductive hearing loss. failure in this process may result in fusion of this joint, as observed in human syndromes such as branchio-oto-renal syndrome or treacher collins syndrome. the malleus and incus first appear as a single united condensation expressing early cartilage markers. in the mouse the first arch derived malleus and incus are formed from a single sox and type ii collagen expressing condensation that later subdivides to give rise to two separate ossicles. we show that the developing incudomalleal joint is characterised by a lack of proliferation and discrete areas of apoptosis.
 the middle ear of mammals is composed of three endochondrial ossicles, the stapes, incus and malleus. joints link the malleus to the incus and the incus to the stapes. in the mouse the first arch derived malleus and incus are formed from a single sox and type ii collagen expressing condensation that later subdivides to give rise to two separate ossicles. in contrast the stapes forms from a separate condensation derived from the second branchial arch. fusion of the malleus and incus is observed in a number of human syndromes and results in conductive hearing loss. understanding how this joint forms during normal development is thus an important step in furthering our understanding of such defects.  we show that the developing incudomalleal joint is characterised by a lack of proliferation and discrete areas of apoptosis. apoptosis has been suggested to aid in the removal of pre-cartilaginous cells from the joint region, allowing for the physical separation of the cartilaginous elements, however, we show that joint initiation is unaffected by blocking apoptosis. there is also no evidence of cell migration out of the presumptive joint region, as observed by labelling of joint and ossicle cells in culture. using type ii collagen lacz reporter mice, however, it is evident that cells in the presumptive joint region remain in place and downregulate cartilage markers.  the malleus and incus first appear as a single united condensation expressing early cartilage markers. the incudomalleal joint region forms by cells in the presumptive joint region switching off cartilage markers and turning on joint markers. failure in this process may result in fusion of this joint, as observed in human syndromes such as branchio-oto-renal syndrome or treacher collins syndrome. 