BACKGROUND
mechanical stimulation plays an important role in skeletal growth and repair reviewed in
<cit>  and, although much less well studied, it is also required for normal skeletal development.
this was initially indicated by observations that infants who experience decreased foetal movement in utero due to neuromuscular disorders present a range of skeletal anomalies including multiple joint fusions, craniofacial abnormalities and thin hypo-mineralised bones
<cit> .
direct evidence that mechanical stimuli generated by embryonic muscle contractions impacts skeletal development comes from a variety of experimental animal models that show similar abnormalities in ossification and joint formation, for example following muscle immobilisation in chick embryos, and in mouse embryos lacking muscle or with reduced or immobile muscle reviewed in
<cit> .
however little is known about the molecular mechanisms through which mechanical stimuli influence cellular events during skeletal development.
the interplay between biophysical stimuli and gene regulation in differentiating cells is emerging as an important phenomenon in multiple developmental systems
<cit> .
a number of different strains of mutant mice have been studied that phenotypically lack limb muscle or show reduced stimuli from muscle contraction during development
<cit> , including splotch  and splotch-delayed , where muscle precursor cells fail to migrate to the developing limbs and no limb muscle forms
<cit> .
common defects in muscle-less and immobilised embryos include abnormal initiation and/or progression of ossification
<cit> , loss of definition of tissue territories in the joint region
<cit>  and altered rudiment morphology
<cit> , associated with reduced local cell proliferation
<cit> .
therefore, mechanical stimuli impact a variety of developmental processes and presumably must influence or integrate with signalling pathways and molecular changes known to guide these events.
one clue to a signalling pathway impacted by mechanical stimulation comes from the work of kahn et al.
<cit>  who showed that canonical wnt signalling is altered in the elbow joint of splotch-delayed embryos.
several regulatory genes have been shown to have dramatically altered expression patterns in reduced mechanical stimuli including, ihh and colx at the site of ossification
<cit>  and bmp <dig>  fgf <dig>  and pthlp at the joint line
<cit> .
whether expression of these genes is directly affected by the mechanical environment or as a more indirect consequence of altered cell behaviour is not known; a genome-wide, open ended screen is required to know more about the spectrum of molecular changes that occur when mechanical stimuli are altered.
gene expression profiling to identify genome-wide changes under altered mechanical environments has been carried out on cells in culture using microarray technology, including osteoblast cell-lines subjected to weightlessness or microgravity conditions
<cit> , chondrocyte-laden constructs and murine cartilage explants to which dynamic compression was applied
<cit>  and chondrocyte cell lines exposed to hydrostatic pressure
<cit> .
gene expression profiling has the potential to uncover hundreds of genes that respond to mechanical stimuli simultaneously ; however no direct analyses of in vivo changes in gene expression during skeletal development following alteration of the mechanical environment have been performed.
this is required to begin to assemble a picture of the molecular landscape impacted by mechanical stimuli in a developmental context.
in this study we analysed the transcriptional changes in the developing humerus and associated joints at theiler stage  23
<cit>    <dig> ) in muscle-less  compared to phenotypically normal littermate controls.
we previously established that the humerus is the most strongly affected rudiment and ts <dig> the earliest time point at which the specific effects on ossification and joint line reduction in the elbow and shoulder regions are detected
<cit> .
we hypothesise that mechanical stimulation of the embryonic skeletal system impacts expression levels of genes implicated in a variety of regulatory pathways and biological processes, as would be expected when an integrated regulatory system is disturbed.
the genes that show altered expression would include direct and indirect targets of mechanical stimulation.
therefore, a genome wide analysis of altered transcript levels is required to indicate the principal molecular mechanisms disturbed and the most likely candidates for direct regulation.
we have used both rna whole transcriptome sequencing analysis  and microarray technology to allow a comprehensive investigation of the altered transcriptome.
microarray analysis is a more established technique
<cit> , but rna-seq offers the potential of greater sensitivity
<cit>  and analysing the same tissues in parallel allows direct comparison of the two assays and integration of the data sets.
we also used rna-seq analysis of the normal developing humerus to explore the transcriptome at this specific stage of development.
the humerus developing in the absence of muscle generated stimulation showed both up and down-regulation of gene expression.
we reveal alteration of genes encoding components and targets of specific signalling pathways, in particular the wnt signalling pathway.
genes associated with cytoskeletal rearrangement and extracellular matrix components are also affected.
this analysis has allowed us to profile genome wide transcriptional changes giving an overview of the molecular processes and pathways most affected and identifying a set of putative direct target genes responding to mechanical stimulation during ossification and joint formation.
RESULTS
transcription profiling of the developing humerus during early ossification and joint formation
rna sequencing of control humeri at ts <dig> gives an insight into the transcriptome at this key stage of development when the rudiment is undergoing early stages of ossification and tissue zones in the joint are being defined .
transcripts from  <dig>  individual genes  were detected in this tissue at this stage.
a minimum of  <dig> transcript reads , with at least one read from each replicate sample, was chosen as the cut off point to reliably indicate reproducible expression across biological replicates
<cit> .
expressed genes were divided into groups according to their relative expression level .
<dig> genes showed the lowest level of expression represented by between  <dig> and  <dig> read counts, while the majority of genes  showed between  <dig> and  <dig> read counts.
only  <dig> genes are in the most highly expressed categories  , representing  <dig> % of expressed genes.
expression levels of selected individual genes are represented in figure
2b.
the most abundantly transcribed gene is col2a <dig>  with a read count of  <dig> , and among the  <dig> genes with read counts of more than  <dig> , there are  <dig> other collagen encoding genes .
in total  <dig> collagen subtype genes are expressed .
the relative levels of expression of genes associated with signalling pathways involved in regulating skeletal development reviewed in
<cit>  are highlighted in figure
2b.
this shows the potential components that can contribute to these signalling pathways at this stage of skeletal development.
for example the hedgehog  pathway is known to play an important role in ossification through the action of ihh , binding to its receptor ptch <dig>  activating smo .
similarly,  <dig> fgf ligand encoding genes and  <dig> fgf receptor encoding genes were detected, highlighting the potential for multiple fgf signalling interactions.
the bmp signalling pathway genes also reveal potential for multiple signalling interactions with  <dig> bmp encoding genes expressed.
bmp <dig> is by far the most highly expressed ; although it is not previously reported in this tissue at this stage in gene expression databases .
the relative expression levels of wnt ligand , wnt receptor , extracellular wnt interactor , intracellular wnt pathway component  and wnt pathway transcription factor  encoding genes are represented.
the detection of previously unreported wnt gene expression in the humerus  opens up new considerations for functional roles, especially as both genes are up-regulated in muscle-less rudiments .
the low density lipoprotein receptor-related genes lrp <dig> and lrp <dig>  which are wnt co-receptors, are most highly expressed among the lrp gene family .
interestingly eight fzd receptor encoding genes are detected.
other genes known to be involved in skeletal development are highly expressed; sox <dig> , runx <dig> , spp <dig>  and mmp <dig> .
identification of differentially expressed genes in muscle-less versus control developing humeri and associated joints
microarray analysis of rna extracted from control and pax3
spd/spd
muscle-less humeri  detected expression of a similar proportion of individual genes on the array;  <dig>  independent genes from the control and  <dig>  from the muscle-less humeri .
comparing hybridisation intensity between control and mutant derived cdnas, using cut off points of at least a 2-fold change and corrected p-value ≤ <dig>
<cit>  for significance across replicates, identified  <dig> independent genes as differentially expressed .
of these,  <dig> genes  were down-regulated and  <dig> genes  were up-regulated .
rna-seq analysis  detected  <dig>  independent genes  in muscle-less humeri, compared to  <dig>  in control tissue .
to determine differential expression, the same cut off points of a corrected p-value ≤ <dig>  and at least a 2-fold change were applied to the rna-seq data-set, identifying  <dig>  genes as de across replicates.
of these,  <dig> genes  were down-regulated and  <dig> genes  were up-regulated in the muscle-less humeri and associated joints compared to that of phenotypically normal littermate control humeri .
the venn diagrams in figure
<dig> represent overlap of de genes identified by the two platforms.
in total  <dig>  independent genes were identified as de in the muscle-less mutant compared to its phenotypic control .
of the  <dig> genes identified by microarray ,  <dig> % of these genes were also represented in the rna-seq data.
rna-seq detected a greater total number of de genes than the microarray .
in addition if the stringency for de gene selection is weakened for the microarray data by moving the cut off point to corrected p-value of ≤ <dig> , the number of genes detected as de in common by the two platforms increases to  <dig> from  <dig> .
this suggests that the rna-seq approach was more sensitive in detecting differential expression.
grouping genes based on the degree of change showed that the spread of fold change values was similar across platforms although there were differences in the number of genes in different categories .
additional file
1: table s <dig> and s <dig> list all de genes and fold change values estimated by microarray and rna-seq.
verification of fold change values for de genes was performed using qrt-pcr .
the de genes chosen  include both down-regulated and up-regulated genes.
the direction and degree of fold changes were similar in all cases for the microarray and the rna-seq.
for all genes analysed there was a good correspondence across all platforms , although greater fold change differences were detected for the down-regulated gene rxrg and the up-regulated gene vstm2a by qrt-pcr.
pax3
spd
/
spd
mutant humeri compared to control humeri revealed by microarray, rna seq and qrt-pcr
#no numerical value for fold change as no transcripts detected in the mutant.
***p ≤  <dig> , **p ≤  <dig> , *p ≤  <dig>  indicates significance of differential expression from qrt-pcr.
p ≤  <dig>  for all microarray and rna-seq differences except spp <dig> in the microarray .
biological interpretation of differentially expressed genes: down-regulated genes are associated with development and differentiation, cytoskeletal architecture and cell signalling
to reveal any enrichment of functionally related genes among the de data sets, two web based tools, david and gostat, were used to analyse gene ontology  term associations
<cit>  .
a significant enrichment  indicates specific biological processes that are affected when mechanical stimuli are reduced.
the strength of the enrichment is indicated by the calculated p-value.
independent analysis of data sets from microarray and rna-seq showed the same enriched groups so analysis of the combined de gene sets is presented .
the individual go terms found to be enriched have been grouped for the purpose of interpretation as indicated in tables
<dig> and
<dig>  analysis of the down-regulated gene set  indicated that genes associated with development and differentiation are most highly enriched .
categories within this group are involved in signal transduction, including genes that encode signalling molecules, receptors, and transcription factors , for example signalling ligands fgf <dig> fgf <dig>  fgf <dig>  fgf <dig> from the fibroblast growth factor signalling pathway; receptors fzd <dig> and rxrg from the wnt and retinoic acid pathways respectively.
<dig> of these genes encode tfs including; barx <dig>  scx, hes <dig>  pitx <dig>  pitx <dig> and tead <dig>  the down-regulation of such signalling pathway component genes also underlies the enrichment of ontology groups related to cell signalling .
1
all go terms are associated with the subontology “biological process” except where indicated under cytoskeleton, where cellular component was also used.
1p-value of enrichment of go terms using david analysis software; similar results were found with gostat.
1
1
1p-value of enrichment of go terms using david analysis software; similar results were found with gostat.
<dig> genes associated with the cytoskeleton were down-regulated in pax3
spd/spd
humeri, including those encoding microfilament, microtubule and intermediate filament components ;  <dig> are directly associated with microfilaments ,  <dig> with microtubules  and  <dig> with intermediate filaments ; other de genes associated with the cytoskeleton encode proteins that interact with myosin, or the extracellular matrix , including integrin and cadherin encoding genes ).
up-regulated genes are associated with cell adhesion, cell signalling and development and differentiation
genes up-regulated in muscle-less humeri revealed by microarray and rna-seq were similarly analysed for enrichment of genes associated with particular biological processes or cellular components, using associated go terms .
for terms within the subontology biological process, the strongest enrichment was for cell adhesion and ecm associated genes .
the genes identified in this category include ecm glycoproteins , ecm structural constituents , cell-adhesion molecules  and calcium-dependent cell adhesion proteins .
this grouping in addition included genes involved in signalling pathways which overlap with the next most enriched terms; cell signalling  and cell-cell communication .
the signalling pathway components identified in this category include: hedgehog , fibroblast growth factor , transforming growth factor , notch  and wnt signalling  and others, including receptors .
other signals identified as up-regulated included c-fos induced gowth factor , hepatocyte growth factor  and insulin-like growth factor .
the gene lists in the next most enriched set, development and differentiation, similarly show large overlap due again to the presence of the signalling pathway genes mentioned above, and also transcription factors foxc <dig>  foxo <dig>  lmx1a, lmx1b.
z.
under the subontology cellular component there was also striking enrichment of extracellular  and membrane associated gene products , including cell adhesion molecules , receptors , cell surface molecules , cadherins , trans-membrane proteins , voltage gated channels  and cell adhesion and extracellular components: alcam, cntn <dig>  epha <dig>  col8a <dig>  col8a <dig>  pappa, pcdh <dig>
signalling pathway analysis of differentially expressed genes
given the strong enrichment of genes associated with signalling pathways and development and differentiation functions , we sorted de genes according to participation in major developmental regulatory pathways .
by far the most strongly impacted cell communication pathway is wnt signalling with  <dig> de genes encoding signalling molecules, receptors, pathway antagonists, known targets or potential targets of the pathway .
from the diagrammatic representation of pathway components shown in figure
<dig>  it is apparent that the encoded products of de genes act either at the cell surface in wnt signal generation/modulation/interpretation or are targets of the pathway.
the genes listed include known targets of the pathway and seven potential target genes , included here due to their similarity to known targets; for example sall1is included because the orthologous gene sall <dig> is a known direct target of the pathway
<cit> .
in general, genes encoding cell surface components of the pathway are up-regulated, including signalling ligands and agonists , and extracellular antagonists , while down-regulated genes identified are more commonly targets of the pathway .
interestingly nine known wnt target genes are up-regulated, including dkk <dig>  rspo <dig>  rspo <dig>  cldn <dig>  grem <dig>  kcnd <dig>  epha <dig> and sfrp <dig>  which encode membrane associated proteins, some of which regulate the wnt pathway .
#no numerical value for fold change as no transcripts detected in the mutant.
t -target, pt -potential target, r -receptor, co-rec -co-receptor, tf-act -transcription factor activator, l -ligand, ant -antagonist, co-act -co-activator.
#no numerical value for fold change as no transcripts detected in the mutant.
co-rec -co-receptor, t -target, l -ligand, l-act- ligand activator, r -receptor, ant -antagonist, co-tf- -co-transcription factor, pl –potential ligand.
table
<dig> lists de genes associated with other developmentally relevant signalling pathways, including the bmp, hedgehog, fibroblast growth factor, hippo and notch signalling pathways.
spatial alteration of gene expression patterns
while microarray and rna-seq analysis provides data on quantitative changes in gene expression levels across the whole developing rudiment, it does not reveal alterations in the spatial distribution of transcripts or give clues to the specific developmental events affected.
we therefore performed in situ hybridisation on control and muscle-less mutant  limb sections at ts <dig> for a selected subset of genes.
three of the genes selected encode components of wnt signalling pathways known to be important in skeletal development: cd <dig>  sfrp <dig> and wnt <dig>  spp <dig> encodes an ecm protein osteopontin which is a prominent component of mineralised matrices of bone and teeth
<cit> .
cd <dig> is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration.
it is a receptor for hyaluronic acid and can interact with other extracellular proteins, such as osteopontin, collagens and matrix metalloprteinases  reviewed in
<cit> .
it is a target gene of the wnt signalling pathway
<cit> .
cd <dig> gene expression is down-regulated  <dig>  fold in muscle-less humeri rna .
the in situ hybridisation analysis reflects this down regulation dramatically with cd <dig> transcripts hardly detectable in either shoulder or elbow joints of muscle-less mutant embryos, compared to the clear joint line restricted expression seen in controls .
sfrp <dig> encodes a secreted protein that acts as a modulator of the wnt signalling pathway, in particular during normal skeletal patterning in developing limbs
<cit> .
normal expression at ts <dig>  can be detected in the elbow and shoulder joints.
the level of up-regulation from microarray and rna-seq analysis is  <dig>   and  <dig>   fold respectively .
this up-regulation was reflected in the intensity of expression seen in mutant sections following in situ hybridisation .
in addition to the increased level of expression a change in the spatial pattern is also evident.
in both the elbow and the shoulder joints expression is expanded, particularly on the ventral aspect of the joint and the staining is unevenly distributed .
a similar expression increase was seen in the phalangeal and carpal joints of the handplate, compared to control .
wnt <dig> is a signalling ligand of the wnt signalling pathway.
expression of this gene has previously indicated its role in joint formation during limb development
<cit> .
control wnt <dig> expression is seen at a low level in the ventral portion of the elbow joint  at ts23; there is also distinct expression in the epidermis, as previously detected
<cit> .
in the mutant there is a high level of expression in the elbow joint region; expression is spread across the whole joint line from ventral to dorsal, more extensive on the dorsal side .
the spp <dig> gene is normally expressed in the hypertrophic zone and adjacent perichondrium
<cit> .
despite the appearance of hypertrophic chondrocytes at the mid-diaphysis of immobile spd embryos , no spp <dig> gene expression is detected in these cells.
there is weak but detectable staining in the perichondrium at the site of the hypertrophic region, but again apparently lower than in control tissue .
expression is also detected in the hypertrophic zone of the ulna  and this is reduced but not absent in the muscle-less mutant ulna , reflecting the reduced phenotypic effect seen in this rudiment
<cit> .
expression is seen only in the perichondrium of the radius in both the control and mutant .
discussion
here we describe the spectrum of genes expressed in the developing humerus at ts <dig>  early in the process of ossification and when territories of differentiating cells are being defined in the developing joint region.
we also use microarray and rna-sequencing to identify genes that are differentially regulated when mechanical stimulation of the developing skeletal rudiment is altered, giving an insight into the genes that respond to mechanical stimuli generated by muscle contractions.
we reveal that the genes altered are highly enriched for genes that regulate development and differentiation, are involved in cytoskeletal rearrangement and components of extracellular matrix including cell adhesion and signalling molecules.
components of multiple signalling pathways important during development are affected, in particular  <dig> components of the wnt signalling pathway.
although it is clear that appropriate mechanical stimulation from in utero muscle contractions is required for normal development of bone and cartilage, we know very little about the molecular mechanisms that incorporate mechanical cues with classical biochemical signalling pathways.
the differentially regulated genes identified here, particularly those associated with signalling pathways and cytoskeletal changes represent a valuable focus for dissecting integrated regulation by biochemical and mechanical signals.
these data represent an important resource that can be utilised to understand the molecular basis of mechanoregulation.
the transcriptome of a developing skeletal rudiment
utilising rna-sequencing technology to reveal the transcriptome in the normal developing humerus and associated joints at ts <dig> provides an insight to the processes that are occurring during this stage of skeletal development when chondrocytes are undergoing hypertrophy, the cartilaginous rudiment is beginning to ossify at the mid diaphysis reviewed in
<cit>  and specific zones within the joint are differentiating reviewed in
<cit> .
this adds a valuable resource to a growing set of data that can be combined to explore skeletal development.
previous transcriptome profiling studies have examined cartilage condensation in the tibia-fibula from e <dig>  to e <dig>
<cit> , ossification of metatarsals
<cit> , in different zones of the growth plate in  <dig> week old mice
<cit>  and in runx2-/- mutant mice
<cit> .
earlier stages of limb development, although not specific to skeletal development, are informed by whole limb bud profiles between e <dig>  and e <dig>
<cit> .
another important resource that has recently become available examines the spatial expression patterns of  <dig>  coding genes and over  <dig> micrornas within the whole mouse embryo at the same developmental stage examined here
<cit> .
this digital transcriptome atlas is a powerful tool  that can be used to examine spatial analysis of specific genes, exploring possible functional associations.
combining these resources gives information on quantitative and spatial expression of individual genes providing the basis to explore regulatory networks active during the development of skeletal rudiments.
several of the findings of the transcriptome analysis are as expected; the most highly expressed genes  include  <dig> collagen encoding genes ; collagens have been shown to be the most abundant structural proteins in cartilage and show characteristic distribution patterns as skeletal rudiments develop
<cit> .
the insulin-like growth factor genes igf <dig> and igf <dig> and their associated receptors ifg2r and igf1r are also highly expressed ; these are reported to play a prominent regulatory role in skeletal development .
similarly, aggrecan  and osteopontin , both involved in skeletal development, are highly expressed .
although much is known about the regulatory network that controls early chondrogenesis and joint formation , open-ended whole transcriptome studies are required to add new information.
centred on regulatory signalling pathways known to be involved in skeletal development; hh, fgf, tgfβ  and wnt; a complete list of components of these pathways expressed in the humerus as ts <dig> was extracted  indicating the potential role players in each of these pathways at this specific stage.
the importance of indian hedgehog  expression in the early prehypertrophic and hypertrophic chondrocytes of cartilage condensations is well established
<cit> .
the full spectrum of possible interacting molecules in hh signalling  include the receptors ptch <dig>  ptch <dig>  smo and transcription factors gli <dig>  gli <dig>  and gli <dig>  in addition to ihh, desert hedgehog  expression was also detected  and, examining the data presented by cameron et al.
confirms that dhh is up-regulated  in e <dig>  fibual and tibual cartilage
<cit> .
dhh has not previously been functionally linked to skeletogenesis but this opens the possibility of regulatory contribution, perhaps co-operating with ihh.
no expression of shh was detected.
similarly, novel components of the fgf, tgbβ and wnt pathways were identified .
the wnt signalling pathway plays a central role during embryonic development and is known to be an important regulator of bone formation and bone remodelling reviewed in
<cit> .
it also plays a pivotal role in joint formation and maintenance, shown through gain and loss of function experiments
<cit> .
the key intracellular mediator of canonical wnt signalling, β-catenin  is the most highly expressed wnt signalling component in the ts <dig> humerus and associated joints.
the most highly expressed wnt ligand is wnt5a, previously associated with expression in joints and perichondrium
<cit>  and proliferating chondrocytes
<cit> .
other highly expressed ligands include wnt9a, wnt5b, wnt <dig>  and wnt <dig>  wnt5b and wnt <dig> expression has been shown in the pre-hypertrophic chondrocytes and wnt <dig>  wnt <dig> and wnt <dig> in the developing joints
<cit> .
high expression of wnt9a could be due to its role in the temporal and spatial regulation of ihh <cit> .
numerous extracellular modulators of the pathway were detected; all five secreted frizzled related protein  genes, dickoff   <dig>   <dig> and  <dig> genes and four r-spondins , indicating a huge potential for pathway modulation.
the most highly expressed antagonist modulators of the pathway were dkk <dig> and sfrp <dig> both of with are detected in joint cells at e <dig>  and e <dig>
<cit> .
the most highly expressed r-spondin agonist of the pathway was rspo <dig>  previously detected in phalanges
<cit> .
identification of mechanoresponsive genes
the identification of differentially expressed genes between humeri from control and muscle-less embryonic limbs allows an investigation of the biological processes and the developmental regulatory signalling pathways that are affected by the removal of mechanical stimulation on skeletogenesis in vivo.
we previously reported that muscleless  mutants display abnormal ossification in the humerus, altered humeral morphogenesis and altered elbow and shoulder joint formation and these effects were first observed at ts23
<cit> .
this was chosen as the point of analysis for differential gene expression because, although limb muscles begin to contract from approximately e <dig>
<cit>  it is uncertain how much stimulation is transmitted to the skeletal rudiments when the developing tendons are at early stages of morphogenesis
<cit> ; the sole indication that the force is functionally transmitted is the mutant phenotype seen at ts <dig>  although analysis at ts <dig> may miss some of the earliest effects, it is relatively early in the response and the earliest time at which it is certain that the system is disturbed.
alteration in expression pattern of some selected candidate genes and pathways was previously revealed
<cit> , but here we carry out the first genome wide study identifying a total of  <dig>  independent genes as differentially expressed: with approximately 60% down-regulated and 40% up-regulated.
the finding of more genes being down-regulated than up-regulated and to a greater extent is consistent with the proposal that mechanical stimuli support the correct differentiation of cells, as observed in the ossification phenotype
<cit> , and for the maintenance of tissue patterning, as seen in the developing joint
<cit> .
go annotation analysis identified specific biological processes that are affected when mechanical stimuli are removed.
this type of analysis has been used previously to interpret biological processes associated with developing skeletal tissue
<cit> .
analysis of the down-regulated de gene set identified genes associated with development and differentiation as the most highly enriched categories, including developmental regulatory signalling pathway molecules and transcription factors.
similarly, analysis of up-regulated de gene sets indicated genes associated with cell signalling and development and differentiation.
de genes were also highly enriched for genes associated with the cytoskeleton.
the cytoskeleton controls cell shape, organelle transport, cell motility and division, and connects the extracellular matrix to internal cell processes reviewed in
<cit> .
it maintains the mechanical integrity of cells and has been implicated in relaying mechanical signals to downstream biochemical responses
<cit> .
this was seen in the embryonic lung where cytoskeletal network inhibitors resulted in altered tissue morphogenesis and conversely when cytoskeletal tension was activated lung development was accelerated reviewed in
<cit> , indicating the dynamic role the cytoskeleton has in morphogenesis.
in chondrocytes the actin microfilaments are predominantly located at the periphery of the cytoplasm
<cit> , tubulin microtubules are uniformly distributed throughout the cytoplasm
<cit>  as are intermediate filaments, connecting the nuclear membrane with the cell periphery
<cit> .
in this study  <dig> genes annotated as cytoskeletal were down-regulated when mechanical stimulation was removed.
these include  <dig> genes directly associated with actin microfilaments,  <dig> with microtubules and  <dig> with intermediate filaments .
the most highly affected group, the filamentous-actin cytoskeleton, has been shown to be involved in articular cartilage chondrocyte mechanotransduction, converting a mechanical stimulus into a biochemical response
<cit> .
other studies have confirmed the involvement of the actin cytoskeleton in cartilage chondrocyte mechanotransduction via manipulation of the actin accessory proteins
<cit> , but there are few reports on the affect of mechanical stimulation on microtubule and intermediate filaments
<cit> .
among the de genes is an actin-binding protein, cofilin <dig> ; cofilin was previously shown to be increased following cyclic mechanical loading of chondrocytes
<cit> .
the identification of cytoskeletal genes down-regulated following the removal of mechanical stimulation indicates that the cytoskeleton is affected, but is this because the mechanical integrity of the cell is altered or because mechanotransduction from the ecm is affected, or perhaps a combination of both?
the finding that ecm and cell adhesion associated genes are also affected further supports changes in mechanotransduction pathways.
the cell-adhesion associated integrins  and cadherins  are down-regulated and these proteins potentially function to physically couple cells to the ecm and play a role in mechanical signal transduction
<cit> .
articular chondrocytes have been shown to express both integrin
<cit>  and non-integrin
<cit>  ecm receptors.
another actin-associated protein identified to be down-regulated is actinin-α <dig> ; this protein also couples the cytoskeleton to the ecm and may be involved in transducing mechanical stimulation.
secreted phosphoprotein  <dig> , previously known as osteopontin , is one of the abundant non-collagenous proteins in bone matrix produced by osteoblasts and osteoclasts reviewed in
<cit> .
spp <dig> binds to hydroxyapatite and is a potent inhibitor of the mineralisation process, inhibiting the growth of bone matrix crystals
<cit> .
spp <dig> is expressed early in bone development, however it was concluded not to be required for normal development of bones as null mice  have no apparent effect on the structure or distribution of cells within bone tissue
<cit> .
however, spp <dig> expression has been shown to be regulated by mechanical stimulation both in vitro and in vivo <cit> .
we found spp <dig> to be down-regulated in the developing humerus at ts <dig> in muscle-less embryos and in situ hybridisation showed a dramatic absence of detectable spp <dig> expression in hypertrophic chondrocytes whereas it is still detectable in the perichondrium , indicating a specific effect on expression in hypertrophic chondrocytes and not a delay in the onset of normal expression.
it was previously shown that opn-/- mice did not suffer bone loss in response to mechanical unloading
<cit> , suggesting that mice lacking spp <dig> could not sense the changes in mechanical stress, thus indicating its potential role in the signal transduction of mechanical stimulation.
it has been suggested that mechanotransduction through spp <dig> is dependent on microfilament integrity, as mechanically stimulated increases in spp <dig> expression was blocked by disruption with cytochalasin-d in osteoblasts
<cit> .
this again highlights the link between an ecm component and the cytoskeleton in a mechanoresponse implicating these components in signal transduction, either directly through the cytoskeleton or through cell adhesion complexes via the cytoskeleton.
an example of a non-integrin ecm component that is down-regulated in the absence of mechanical stimulation is cd <dig>  a target gene of the wnt signalling pathway
<cit> , encoding a single-pass membrane glycoprotein that binds proteoglycan and hyaluronan  to produce a pericellular matrix surrounding chondrocytes reviewed in
<cit> .
cd <dig> has been implicated in joint cavitation through interaction with ha
<cit> .
we previously showed loss of expression of the cd <dig> gene in the interzone of the forming knee joints of immobilised chick embryos
<cit> , one of a number of gene expression patterns reflecting a general loss of organisation of differentiating tissue territories, and here we show a similar effect on cd <dig> expression in the elbow and shoulder joint of muscle-less mouse limbs, where we previously showed a similar loss of tissue organisation
<cit> .
the very restricted expression of cd <dig> in the interzone of forming joints in control embryos at ts <dig> is barely detectable in muscle-less mutants .
as well as the gene being sensitive to mechanical stimulation, as an integral part of the ecm and a regulator of joint formation, the gene product may also be an important mediator of mechanical stimuli.
the link between cd <dig> and the wnt signalling pathway highlights perhaps the most striking finding of this analysis; the altered expression of  <dig> genes implicated in the wnt signalling pathway .
canonical wnt signalling has been shown to be involved in maintaining joint integrity
<cit>  and is disturbed in the joints of muscle-less mouse embryos
<cit> .
wnt signalling might also be involved in co-ordinating ossification and joint development; both processes altered in muscle-less embryos
<cit> .
non canonical signalling has also been implicated in planar cell polarity during growth plate regulation
<cit> .
there are also previous indications that the wnt pathway is responsive to mechanical stimulation in mesenchymal stem cells
<cit>  in mature bone in vivo <cit>  and in response to injury of articular cartilage
<cit> .
here, genes encoding four wnt ligands are up-regulated in muscle-less embryos and in the case of wnt <dig> we show specific up-regulation in the elbow and shoulder joint region .
two of the up-regulated genes  have not previously been associated with skeletal development.
the wnt signalling antagonist sfrp <dig> is also up-regulated specifically in the joint region .
the majority of known wnt target genes affected are down-regulated , perhaps due to increased expression of negative regulators sfrp <dig> and dkk <dig> and down-regulation of the fzd <dig> receptor; however ligands and agonists rspo <dig> and rspo <dig> are up-regulated as are some target genes indicating effects at multiple levels of regulation of the pathway.
it is interesting that a number of the up-regulated targets feedback as negative regulators of the pathway .
it is now important to functionally test the mechanisms linking mechanical stimulation with wnt signalling.
this work provides sets of candidate genes to use in functional assays to excavate this important link.
understanding how mechanical stimuli influence the wnt signalling pathway would be a major step forward in understanding how mechanical cues work together with classical molecular positional information to guide spatially appropriate tissue differentiation and provide indications of how conditions can be effectively recreated in vitro to guide stem cell differentiation.
in situ hybridisation analysis showed altered gene expression of  <dig> wnt pathway genes in the developing shoulder and elbow joints; one down regulated  and two upregulated  .
changes in the spatial restriction of sfrp <dig> and wnt <dig> expression were also seen.
this does not represent a delay in normal expression in the mutant because the altered pattern is not reminiscent of earlier stages
<cit>  and the changes are consistent with altered patterning of the tissue territories in the forming joint and the fusion phenotype seen in the mutant, with cartilage forming across the joint at later stages
<cit> .
in this study differential expression in developing skeletal rudiments is documented in the absence of limb muscle; this will include genes that respond to lack of mechanical stimulation but perhaps also as a paracrine response to adjacent muscle cells.
we know that phenotypic effects on ossification and joint formation are due to the lack of mechanical stimulation rather than physical absence of muscle cells because phenotypic analysis of a range of mouse mutants where muscle is immobile
<cit> , reduced
<cit>  or absent
<cit>  have similar effects and we see similar effects in immobilised chick embryos
<cit> .
therefore, although some of the genes identified here may respond to lack of adjacent muscle tissue, many must underlie the phenotypic effects seen in response to lack of mechanical stimulation.
this is further supported by the overlap of some of the genes identified here and in skeletal cells in culture or adult tissues, in response to mechanical stimulation
<cit> .
another important limitation in this work is the possibility that a proportion of the down-regulated genes may be due to contamination of the dissected control humeri with adjacent mesenchyme/muscle, since this is being compared to tissue from muscle-less embryos.
although care was taken with the dissections, it is impossible to be sure that all muscle tissue was eliminated from the control.
the down-regulated gene set also showed enrichment for muscle associated genes , consistent with possible contamination of the dissected control humeri by neighbouring muscle.
to inform this we also sequenced the transcriptome of mesenchyme adjacent to the humerus of control embryos at ts <dig> and compared it to the transcriptome of control humeri.
we then cross referenced this to the down-regulated gene set in control versus muscle-less humeri , noting any genes enriched more than  <dig> fold in mesenchyme compared to control humeri; these are indicated in column  <dig> of additional file
1: table s <dig>  it is possible that these genes are involved in both cartilage and muscle development so no genes have been removed from the data set, however, de genes also showing higher expression in mesenchyme compared to control humeri must be treated with caution with respect to a skeletal specific response to mechanical stimulation.
such genes have not been prioritised in any of our subsequent exploration of candidate mechanosensitive genes.
the developing humerus at ts <dig> constitutes different cell and tissue populations at different stages of differentiation including the joint region, the perichondrium and the organised zones within the cartilage rudiment.
therefore the experimental design employed here will capture genes associated with different cells types at different stages of differentiation.
it will now be important to sort out which cells and tissues have altered expression of specific genes.
this can be addressed for a sub set of genes by in situ hybridisation, with an initial analysis of  <dig> genes presented in figure
<dig>  it can be addressed in a high throughput manner by isolating specific cell populations using laser microdissection from tissue sections , purification of rna and quantitative rt-pcr gene expression profiling, comparing control and mutant tissue from,, for example the hypertrophic, prehypertrophic or the elbow joint region alone.
we used both rna-sequencing  and microarray  technologies in parallel to determine differential expression.
microarray technology has been utilised to determine expression of chondrogenic and osteogenic genes from developing whole tissues
<cit> , and from in vitro differentiation procedures
<cit> .
the use of rna-seq technology to describe the transcriptome is more recent
<cit> .
previous direct comparisons between microarray and rna sequencing-based approaches to reveal alterations in gene expression between tissues reported that rna-seq identified more de genes
<cit> .
we also found that rna-seq is more sensitive in reproducibly detecting alterations in gene expression, detecting more genes altered at lower quantitative levels .
this was further emphasised by reducing the stringency of the statistical analysis to p ≤  <dig> , which increased the number of genes detected by microarray specifically .
an example of the importance of the increased sensitivity and reproducibility of rna-seq is shown by the spp <dig> gene which did not show statistical significance by microarray but has been verified by qrt-pcr and in situ hybridisation .
the larger dynamic range
<cit>  and higher reproducibility across replicates
<cit>  has also been found in other studies.
CONCLUSIONS
this study examines the set of genes active at a key stage of skeletal development  and reveals the genes that are differentially regulated in the developing humerus when skeletal muscle is absent.
since we previously showed that the lack of muscle contractions leads to common phenotypic defects in both ossification and joint formation in several chick and mouse models, this provides an insight into the genome wide alterations in gene transcription that take place when the mechanical environment is altered.
given the importance of appropriate mechanical stimulation generated by embryo movement on skeletal development we postulated that mechanical stimuli must integrate with biochemical cell signalling pathways known to be essential for normal development.
we show that multiple signalling pathways are affected, with components of the wnt signalling pathway most strongly disturbed including  <dig> wnt ligands and both down-regulation and up-regulation of target genes.
down-regulated genes include cd <dig>  dll <dig> and fgf <dig> which are involved in further cellular interactions during joint formation or feed into other important cell communication events.
among the up-regulated wnt targets are several genes that feed back into the wnt pathway itself as antagonists  or agonists .
this finding, together with alteration of cytoskeletal components, indicates the biological processes involved in integrating biophysical stimuli during cell differentiation and patterning.
understanding the mechanistic basis for how developing cells interpret and respond to biophysical cues is a major challenge, relevant to all developing systems, and will impact our ability to control differentiation of progenitor cells for regenerative therapies.
this work is an early step in unravelling the mechanistic basis of biophysical regulation of skeletal development and provides a focus for future studies.

 mechanical stimulation plays an important role in skeletal growth and repair reviewed in and, although much less well studied, it is also required for normal skeletal development. this was initially indicated by observations that infants who experience decreased foetal movement in utero due to neuromuscular disorders present a range of skeletal anomalies including multiple joint fusions, craniofacial abnormalities and thin hypo-mineralised bones. direct evidence that mechanical stimuli generated by embryonic muscle contractions impacts skeletal development comes from a variety of experimental animal models that show similar abnormalities in ossification and joint formation, for example following muscle immobilisation in chick embryos, and in mouse embryos lacking muscle or with reduced or immobile muscle reviewed in. however little is known about the molecular mechanisms through which mechanical stimuli influence cellular events during skeletal development. the interplay between biophysical stimuli and gene regulation in differentiating cells is emerging as an important phenomenon in multiple developmental systems. a number of different strains of mutant mice have been studied that phenotypically lack limb muscle or show reduced stimuli from muscle contraction during development, including splotch and splotch-delayed, where muscle precursor cells fail to migrate to the developing limbs and no limb muscle forms. common defects in muscle-less and immobilised embryos include abnormal initiation and/or progression of ossification, loss of definition of tissue territories in the joint region and altered rudiment morphology, associated with reduced local cell proliferation. therefore, mechanical stimuli impact a variety of developmental processes and presumably must influence or integrate with signalling pathways and molecular changes known to guide these events. one clue to a signalling pathway impacted by mechanical stimulation comes from the work of kahn et al. who showed that canonical wnt signalling is altered in the elbow joint of splotch-delayed embryos. several regulatory genes have been shown to have dramatically altered expression patterns in reduced mechanical stimuli including, ihh and colx at the site of ossification and bmp fgf and pthlp at the joint line. whether expression of these genes is directly affected by the mechanical environment or as a more indirect consequence of altered cell behaviour is not known; a genome-wide, open ended screen is required to know more about the spectrum of molecular changes that occur when mechanical stimuli are altered. gene expression profiling to identify genome-wide changes under altered mechanical environments has been carried out on cells in culture using microarray technology, including osteoblast cell-lines subjected to weightlessness or microgravity conditions, chondrocyte-laden constructs and murine cartilage explants to which dynamic compression was applied and chondrocyte cell lines exposed to hydrostatic pressure. gene expression profiling has the potential to uncover hundreds of genes that respond to mechanical stimuli simultaneously; however no direct analyses of in vivo changes in gene expression during skeletal development following alteration of the mechanical environment have been performed. this is required to begin to assemble a picture of the molecular landscape impacted by mechanical stimuli in a developmental context. in this study we analysed the transcriptional changes in the developing humerus and associated joints at theiler stage 23 ) in muscle-less compared to phenotypically normal littermate controls. we previously established that the humerus is the most strongly affected rudiment and ts the earliest time point at which the specific effects on ossification and joint line reduction in the elbow and shoulder regions are detected. we hypothesise that mechanical stimulation of the embryonic skeletal system impacts expression levels of genes implicated in a variety of regulatory pathways and biological processes, as would be expected when an integrated regulatory system is disturbed. the genes that show altered expression would include direct and indirect targets of mechanical stimulation. therefore, a genome wide analysis of altered transcript levels is required to indicate the principal molecular mechanisms disturbed and the most likely candidates for direct regulation. we have used both rna whole transcriptome sequencing analysis and microarray technology to allow a comprehensive investigation of the altered transcriptome. microarray analysis is a more established technique, but rna-seq offers the potential of greater sensitivity and analysing the same tissues in parallel allows direct comparison of the two assays and integration of the data sets. we also used rna-seq analysis of the normal developing humerus to explore the transcriptome at this specific stage of development. the humerus developing in the absence of muscle generated stimulation showed both up and down-regulation of gene expression. we reveal alteration of genes encoding components and targets of specific signalling pathways, in particular the wnt signalling pathway. genes associated with cytoskeletal rearrangement and extracellular matrix components are also affected. this analysis has allowed us to profile genome wide transcriptional changes giving an overview of the molecular processes and pathways most affected and identifying a set of putative direct target genes responding to mechanical stimulation during ossification and joint formation.  transcription profiling of the developing humerus during early ossification and joint formation rna sequencing of control humeri at ts gives an insight into the transcriptome at this key stage of development when the rudiment is undergoing early stages of ossification and tissue zones in the joint are being defined. transcripts from individual genes were detected in this tissue at this stage. a minimum of transcript reads, with at least one read from each replicate sample, was chosen as the cut off point to reliably indicate reproducible expression across biological replicates. expressed genes were divided into groups according to their relative expression level. genes showed the lowest level of expression represented by between and read counts, while the majority of genes showed between and read counts. only genes are in the most highly expressed categories, representing % of expressed genes. expression levels of selected individual genes are represented in figure  2b. the most abundantly transcribed gene is col2a with a read count of, and among the genes with read counts of more than, there are other collagen encoding genes. in total collagen subtype genes are expressed. the relative levels of expression of genes associated with signalling pathways involved in regulating skeletal development reviewed in are highlighted in figure  2b. this shows the potential components that can contribute to these signalling pathways at this stage of skeletal development. for example the hedgehog pathway is known to play an important role in ossification through the action of ihh, binding to its receptor ptch activating smo. similarly, fgf ligand encoding genes and fgf receptor encoding genes were detected, highlighting the potential for multiple fgf signalling interactions. the bmp signalling pathway genes also reveal potential for multiple signalling interactions with bmp encoding genes expressed. bmp is by far the most highly expressed; although it is not previously reported in this tissue at this stage in gene expression databases. the relative expression levels of wnt ligand, wnt receptor, extracellular wnt interactor, intracellular wnt pathway component and wnt pathway transcription factor encoding genes are represented. the detection of previously unreported wnt gene expression in the humerus opens up new considerations for functional roles, especially as both genes are up-regulated in muscle-less rudiments. the low density lipoprotein receptor-related genes lrp and lrp which are wnt co-receptors, are most highly expressed among the lrp gene family. interestingly eight fzd receptor encoding genes are detected. other genes known to be involved in skeletal development are highly expressed; sox, runx, spp and mmp. identification of differentially expressed genes in muscle-less versus control developing humeri and associated joints microarray analysis of rna extracted from control and pax3 spd/spd muscle-less humeri detected expression of a similar proportion of individual genes on the array; independent genes from the control and from the muscle-less humeri. comparing hybridisation intensity between control and mutant derived cdnas, using cut off points of at least a 2-fold change and corrected p-value ≤ for significance across replicates, identified independent genes as differentially expressed. of these, genes were down-regulated and genes were up-regulated. rna-seq analysis detected independent genes in muscle-less humeri, compared to in control tissue. to determine differential expression, the same cut off points of a corrected p-value ≤ and at least a 2-fold change were applied to the rna-seq data-set, identifying genes as de across replicates. of these, genes were down-regulated and genes were up-regulated in the muscle-less humeri and associated joints compared to that of phenotypically normal littermate control humeri. the venn diagrams in figure  represent overlap of de genes identified by the two platforms. in total independent genes were identified as de in the muscle-less mutant compared to its phenotypic control. of the genes identified by microarray, % of these genes were also represented in the rna-seq data. rna-seq detected a greater total number of de genes than the microarray. in addition if the stringency for de gene selection is weakened for the microarray data by moving the cut off point to corrected p-value of ≤, the number of genes detected as de in common by the two platforms increases to from. this suggests that the rna-seq approach was more sensitive in detecting differential expression. grouping genes based on the degree of change showed that the spread of fold change values was similar across platforms although there were differences in the number of genes in different categories. additional file 1: table s and s list all de genes and fold change values estimated by microarray and rna-seq. verification of fold change values for de genes was performed using qrt-pcr. the de genes chosen include both down-regulated and up-regulated genes. the direction and degree of fold changes were similar in all cases for the microarray and the rna-seq. for all genes analysed there was a good correspondence across all platforms, although greater fold change differences were detected for the down-regulated gene rxrg and the up-regulated gene vstm2a by qrt-pcr. pax3 spd / spd mutant humeri compared to control humeri revealed by microarray, rna seq and qrt-pcr #no numerical value for fold change as no transcripts detected in the mutant. ***p ≤ , **p ≤ , *p ≤  indicates significance of differential expression from qrt-pcr. p ≤  for all microarray and rna-seq differences except spp in the microarray. biological interpretation of differentially expressed genes: down-regulated genes are associated with development and differentiation, cytoskeletal architecture and cell signalling to reveal any enrichment of functionally related genes among the de data sets, two web based tools, david and gostat, were used to analyse gene ontology term associations. a significant enrichment indicates specific biological processes that are affected when mechanical stimuli are reduced. the strength of the enrichment is indicated by the calculated p-value. independent analysis of data sets from microarray and rna-seq showed the same enriched groups so analysis of the combined de gene sets is presented. the individual go terms found to be enriched have been grouped for the purpose of interpretation as indicated in tables  and analysis of the down-regulated gene set indicated that genes associated with development and differentiation are most highly enriched. categories within this group are involved in signal transduction, including genes that encode signalling molecules, receptors, and transcription factors, for example signalling ligands fgf fgf fgf fgf from the fibroblast growth factor signalling pathway; receptors fzd and rxrg from the wnt and retinoic acid pathways respectively. of these genes encode tfs including; barx scx, hes pitx pitx and tead the down-regulation of such signalling pathway component genes also underlies the enrichment of ontology groups related to cell signalling. 1 all go terms are associated with the subontology “biological process” except where indicated under cytoskeleton, where cellular component was also used. 1p-value of enrichment of go terms using david analysis software; similar results were found with gostat. 1 1 1p-value of enrichment of go terms using david analysis software; similar results were found with gostat. genes associated with the cytoskeleton were down-regulated in pax3 spd/spd humeri, including those encoding microfilament, microtubule and intermediate filament components; are directly associated with microfilaments, with microtubules and with intermediate filaments; other de genes associated with the cytoskeleton encode proteins that interact with myosin, or the extracellular matrix, including integrin and cadherin encoding genes ). up-regulated genes are associated with cell adhesion, cell signalling and development and differentiation genes up-regulated in muscle-less humeri revealed by microarray and rna-seq were similarly analysed for enrichment of genes associated with particular biological processes or cellular components, using associated go terms. for terms within the subontology biological process, the strongest enrichment was for cell adhesion and ecm associated genes. the genes identified in this category include ecm glycoproteins, ecm structural constituents, cell-adhesion molecules and calcium-dependent cell adhesion proteins. this grouping in addition included genes involved in signalling pathways which overlap with the next most enriched terms; cell signalling and cell-cell communication. the signalling pathway components identified in this category include: hedgehog, fibroblast growth factor, transforming growth factor, notch and wnt signalling and others, including receptors. other signals identified as up-regulated included c-fos induced gowth factor, hepatocyte growth factor and insulin-like growth factor. the gene lists in the next most enriched set, development and differentiation, similarly show large overlap due again to the presence of the signalling pathway genes mentioned above, and also transcription factors foxc foxo lmx1a, lmx1b. z. under the subontology cellular component there was also striking enrichment of extracellular and membrane associated gene products, including cell adhesion molecules, receptors, cell surface molecules, cadherins, trans-membrane proteins, voltage gated channels and cell adhesion and extracellular components: alcam, cntn epha col8a col8a pappa, pcdh signalling pathway analysis of differentially expressed genes given the strong enrichment of genes associated with signalling pathways and development and differentiation functions, we sorted de genes according to participation in major developmental regulatory pathways. by far the most strongly impacted cell communication pathway is wnt signalling with de genes encoding signalling molecules, receptors, pathway antagonists, known targets or potential targets of the pathway. from the diagrammatic representation of pathway components shown in figure  it is apparent that the encoded products of de genes act either at the cell surface in wnt signal generation/modulation/interpretation or are targets of the pathway. the genes listed include known targets of the pathway and seven potential target genes, included here due to their similarity to known targets; for example sall1is included because the orthologous gene sall is a known direct target of the pathway. in general, genes encoding cell surface components of the pathway are up-regulated, including signalling ligands and agonists, and extracellular antagonists, while down-regulated genes identified are more commonly targets of the pathway. interestingly nine known wnt target genes are up-regulated, including dkk rspo rspo cldn grem kcnd epha and sfrp which encode membrane associated proteins, some of which regulate the wnt pathway. #no numerical value for fold change as no transcripts detected in the mutant. t -target, pt -potential target, r -receptor, co-rec -co-receptor, tf-act -transcription factor activator, l -ligand, ant -antagonist, co-act -co-activator. #no numerical value for fold change as no transcripts detected in the mutant. co-rec -co-receptor, t -target, l -ligand, l-act- ligand activator, r -receptor, ant -antagonist, co-tf- -co-transcription factor, pl –potential ligand. table  lists de genes associated with other developmentally relevant signalling pathways, including the bmp, hedgehog, fibroblast growth factor, hippo and notch signalling pathways. spatial alteration of gene expression patterns while microarray and rna-seq analysis provides data on quantitative changes in gene expression levels across the whole developing rudiment, it does not reveal alterations in the spatial distribution of transcripts or give clues to the specific developmental events affected. we therefore performed in situ hybridisation on control and muscle-less mutant limb sections at ts for a selected subset of genes. three of the genes selected encode components of wnt signalling pathways known to be important in skeletal development: cd sfrp and wnt spp encodes an ecm protein osteopontin which is a prominent component of mineralised matrices of bone and teeth. cd is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. it is a receptor for hyaluronic acid and can interact with other extracellular proteins, such as osteopontin, collagens and matrix metalloprteinases reviewed in. it is a target gene of the wnt signalling pathway. cd gene expression is down-regulated fold in muscle-less humeri rna. the in situ hybridisation analysis reflects this down regulation dramatically with cd transcripts hardly detectable in either shoulder or elbow joints of muscle-less mutant embryos, compared to the clear joint line restricted expression seen in controls. sfrp encodes a secreted protein that acts as a modulator of the wnt signalling pathway, in particular during normal skeletal patterning in developing limbs. normal expression at ts can be detected in the elbow and shoulder joints. the level of up-regulation from microarray and rna-seq analysis is and fold respectively. this up-regulation was reflected in the intensity of expression seen in mutant sections following in situ hybridisation. in addition to the increased level of expression a change in the spatial pattern is also evident. in both the elbow and the shoulder joints expression is expanded, particularly on the ventral aspect of the joint and the staining is unevenly distributed. a similar expression increase was seen in the phalangeal and carpal joints of the handplate, compared to control. wnt is a signalling ligand of the wnt signalling pathway. expression of this gene has previously indicated its role in joint formation during limb development. control wnt expression is seen at a low level in the ventral portion of the elbow joint at ts23; there is also distinct expression in the epidermis, as previously detected. in the mutant there is a high level of expression in the elbow joint region; expression is spread across the whole joint line from ventral to dorsal, more extensive on the dorsal side. the spp gene is normally expressed in the hypertrophic zone and adjacent perichondrium. despite the appearance of hypertrophic chondrocytes at the mid-diaphysis of immobile spd embryos, no spp gene expression is detected in these cells. there is weak but detectable staining in the perichondrium at the site of the hypertrophic region, but again apparently lower than in control tissue. expression is also detected in the hypertrophic zone of the ulna and this is reduced but not absent in the muscle-less mutant ulna, reflecting the reduced phenotypic effect seen in this rudiment. expression is seen only in the perichondrium of the radius in both the control and mutant. discussion here we describe the spectrum of genes expressed in the developing humerus at ts early in the process of ossification and when territories of differentiating cells are being defined in the developing joint region. we also use microarray and rna-sequencing to identify genes that are differentially regulated when mechanical stimulation of the developing skeletal rudiment is altered, giving an insight into the genes that respond to mechanical stimuli generated by muscle contractions. we reveal that the genes altered are highly enriched for genes that regulate development and differentiation, are involved in cytoskeletal rearrangement and components of extracellular matrix including cell adhesion and signalling molecules. components of multiple signalling pathways important during development are affected, in particular components of the wnt signalling pathway. although it is clear that appropriate mechanical stimulation from in utero muscle contractions is required for normal development of bone and cartilage, we know very little about the molecular mechanisms that incorporate mechanical cues with classical biochemical signalling pathways. the differentially regulated genes identified here, particularly those associated with signalling pathways and cytoskeletal changes represent a valuable focus for dissecting integrated regulation by biochemical and mechanical signals. these data represent an important resource that can be utilised to understand the molecular basis of mechanoregulation. the transcriptome of a developing skeletal rudiment utilising rna-sequencing technology to reveal the transcriptome in the normal developing humerus and associated joints at ts provides an insight to the processes that are occurring during this stage of skeletal development when chondrocytes are undergoing hypertrophy, the cartilaginous rudiment is beginning to ossify at the mid diaphysis reviewed in and specific zones within the joint are differentiating reviewed in. this adds a valuable resource to a growing set of data that can be combined to explore skeletal development. previous transcriptome profiling studies have examined cartilage condensation in the tibia-fibula from e to e, ossification of metatarsals, in different zones of the growth plate in week old mice and in runx2-/- mutant mice. earlier stages of limb development, although not specific to skeletal development, are informed by whole limb bud profiles between e and e. another important resource that has recently become available examines the spatial expression patterns of coding genes and over micrornas within the whole mouse embryo at the same developmental stage examined here. this digital transcriptome atlas is a powerful tool that can be used to examine spatial analysis of specific genes, exploring possible functional associations. combining these resources gives information on quantitative and spatial expression of individual genes providing the basis to explore regulatory networks active during the development of skeletal rudiments. several of the findings of the transcriptome analysis are as expected; the most highly expressed genes include collagen encoding genes; collagens have been shown to be the most abundant structural proteins in cartilage and show characteristic distribution patterns as skeletal rudiments develop. the insulin-like growth factor genes igf and igf and their associated receptors ifg2r and igf1r are also highly expressed; these are reported to play a prominent regulatory role in skeletal development. similarly, aggrecan and osteopontin, both involved in skeletal development, are highly expressed. although much is known about the regulatory network that controls early chondrogenesis and joint formation, open-ended whole transcriptome studies are required to add new information. centred on regulatory signalling pathways known to be involved in skeletal development; hh, fgf, tgfβ and wnt; a complete list of components of these pathways expressed in the humerus as ts was extracted indicating the potential role players in each of these pathways at this specific stage. the importance of indian hedgehog expression in the early prehypertrophic and hypertrophic chondrocytes of cartilage condensations is well established. the full spectrum of possible interacting molecules in hh signalling include the receptors ptch ptch smo and transcription factors gli gli and gli in addition to ihh, desert hedgehog expression was also detected and, examining the data presented by cameron et al. confirms that dhh is up-regulated in e fibual and tibual cartilage. dhh has not previously been functionally linked to skeletogenesis but this opens the possibility of regulatory contribution, perhaps co-operating with ihh. no expression of shh was detected. similarly, novel components of the fgf, tgbβ and wnt pathways were identified. the wnt signalling pathway plays a central role during embryonic development and is known to be an important regulator of bone formation and bone remodelling reviewed in. it also plays a pivotal role in joint formation and maintenance, shown through gain and loss of function experiments. the key intracellular mediator of canonical wnt signalling, β-catenin is the most highly expressed wnt signalling component in the ts humerus and associated joints. the most highly expressed wnt ligand is wnt5a, previously associated with expression in joints and perichondrium and proliferating chondrocytes. other highly expressed ligands include wnt9a, wnt5b, wnt and wnt wnt5b and wnt expression has been shown in the pre-hypertrophic chondrocytes and wnt wnt and wnt in the developing joints. high expression of wnt9a could be due to its role in the temporal and spatial regulation of ihh. numerous extracellular modulators of the pathway were detected; all five secreted frizzled related protein genes, dickoff and genes and four r-spondins, indicating a huge potential for pathway modulation. the most highly expressed antagonist modulators of the pathway were dkk and sfrp both of with are detected in joint cells at e and e. the most highly expressed r-spondin agonist of the pathway was rspo previously detected in phalanges. identification of mechanoresponsive genes the identification of differentially expressed genes between humeri from control and muscle-less embryonic limbs allows an investigation of the biological processes and the developmental regulatory signalling pathways that are affected by the removal of mechanical stimulation on skeletogenesis in vivo. we previously reported that muscleless mutants display abnormal ossification in the humerus, altered humeral morphogenesis and altered elbow and shoulder joint formation and these effects were first observed at ts23. this was chosen as the point of analysis for differential gene expression because, although limb muscles begin to contract from approximately e it is uncertain how much stimulation is transmitted to the skeletal rudiments when the developing tendons are at early stages of morphogenesis; the sole indication that the force is functionally transmitted is the mutant phenotype seen at ts although analysis at ts may miss some of the earliest effects, it is relatively early in the response and the earliest time at which it is certain that the system is disturbed. alteration in expression pattern of some selected candidate genes and pathways was previously revealed, but here we carry out the first genome wide study identifying a total of independent genes as differentially expressed: with approximately 60% down-regulated and 40% up-regulated. the finding of more genes being down-regulated than up-regulated and to a greater extent is consistent with the proposal that mechanical stimuli support the correct differentiation of cells, as observed in the ossification phenotype, and for the maintenance of tissue patterning, as seen in the developing joint. go annotation analysis identified specific biological processes that are affected when mechanical stimuli are removed. this type of analysis has been used previously to interpret biological processes associated with developing skeletal tissue. analysis of the down-regulated de gene set identified genes associated with development and differentiation as the most highly enriched categories, including developmental regulatory signalling pathway molecules and transcription factors. similarly, analysis of up-regulated de gene sets indicated genes associated with cell signalling and development and differentiation. de genes were also highly enriched for genes associated with the cytoskeleton. the cytoskeleton controls cell shape, organelle transport, cell motility and division, and connects the extracellular matrix to internal cell processes reviewed in. it maintains the mechanical integrity of cells and has been implicated in relaying mechanical signals to downstream biochemical responses. this was seen in the embryonic lung where cytoskeletal network inhibitors resulted in altered tissue morphogenesis and conversely when cytoskeletal tension was activated lung development was accelerated reviewed in, indicating the dynamic role the cytoskeleton has in morphogenesis. in chondrocytes the actin microfilaments are predominantly located at the periphery of the cytoplasm, tubulin microtubules are uniformly distributed throughout the cytoplasm as are intermediate filaments, connecting the nuclear membrane with the cell periphery. in this study genes annotated as cytoskeletal were down-regulated when mechanical stimulation was removed. these include genes directly associated with actin microfilaments, with microtubules and with intermediate filaments. the most highly affected group, the filamentous-actin cytoskeleton, has been shown to be involved in articular cartilage chondrocyte mechanotransduction, converting a mechanical stimulus into a biochemical response. other studies have confirmed the involvement of the actin cytoskeleton in cartilage chondrocyte mechanotransduction via manipulation of the actin accessory proteins, but there are few reports on the affect of mechanical stimulation on microtubule and intermediate filaments. among the de genes is an actin-binding protein, cofilin; cofilin was previously shown to be increased following cyclic mechanical loading of chondrocytes. the identification of cytoskeletal genes down-regulated following the removal of mechanical stimulation indicates that the cytoskeleton is affected, but is this because the mechanical integrity of the cell is altered or because mechanotransduction from the ecm is affected, or perhaps a combination of both? the finding that ecm and cell adhesion associated genes are also affected further supports changes in mechanotransduction pathways. the cell-adhesion associated integrins and cadherins are down-regulated and these proteins potentially function to physically couple cells to the ecm and play a role in mechanical signal transduction. articular chondrocytes have been shown to express both integrin and non-integrin ecm receptors. another actin-associated protein identified to be down-regulated is actinin-α; this protein also couples the cytoskeleton to the ecm and may be involved in transducing mechanical stimulation. secreted phosphoprotein, previously known as osteopontin, is one of the abundant non-collagenous proteins in bone matrix produced by osteoblasts and osteoclasts reviewed in. spp binds to hydroxyapatite and is a potent inhibitor of the mineralisation process, inhibiting the growth of bone matrix crystals. spp is expressed early in bone development, however it was concluded not to be required for normal development of bones as null mice have no apparent effect on the structure or distribution of cells within bone tissue. however, spp expression has been shown to be regulated by mechanical stimulation both in vitro and in vivo. we found spp to be down-regulated in the developing humerus at ts in muscle-less embryos and in situ hybridisation showed a dramatic absence of detectable spp expression in hypertrophic chondrocytes whereas it is still detectable in the perichondrium, indicating a specific effect on expression in hypertrophic chondrocytes and not a delay in the onset of normal expression. it was previously shown that opn-/- mice did not suffer bone loss in response to mechanical unloading, suggesting that mice lacking spp could not sense the changes in mechanical stress, thus indicating its potential role in the signal transduction of mechanical stimulation. it has been suggested that mechanotransduction through spp is dependent on microfilament integrity, as mechanically stimulated increases in spp expression was blocked by disruption with cytochalasin-d in osteoblasts. this again highlights the link between an ecm component and the cytoskeleton in a mechanoresponse implicating these components in signal transduction, either directly through the cytoskeleton or through cell adhesion complexes via the cytoskeleton. an example of a non-integrin ecm component that is down-regulated in the absence of mechanical stimulation is cd a target gene of the wnt signalling pathway, encoding a single-pass membrane glycoprotein that binds proteoglycan and hyaluronan to produce a pericellular matrix surrounding chondrocytes reviewed in. cd has been implicated in joint cavitation through interaction with ha. we previously showed loss of expression of the cd gene in the interzone of the forming knee joints of immobilised chick embryos, one of a number of gene expression patterns reflecting a general loss of organisation of differentiating tissue territories, and here we show a similar effect on cd expression in the elbow and shoulder joint of muscle-less mouse limbs, where we previously showed a similar loss of tissue organisation. the very restricted expression of cd in the interzone of forming joints in control embryos at ts is barely detectable in muscle-less mutants. as well as the gene being sensitive to mechanical stimulation, as an integral part of the ecm and a regulator of joint formation, the gene product may also be an important mediator of mechanical stimuli. the link between cd and the wnt signalling pathway highlights perhaps the most striking finding of this analysis; the altered expression of genes implicated in the wnt signalling pathway. canonical wnt signalling has been shown to be involved in maintaining joint integrity and is disturbed in the joints of muscle-less mouse embryos. wnt signalling might also be involved in co-ordinating ossification and joint development; both processes altered in muscle-less embryos. non canonical signalling has also been implicated in planar cell polarity during growth plate regulation. there are also previous indications that the wnt pathway is responsive to mechanical stimulation in mesenchymal stem cells in mature bone in vivo and in response to injury of articular cartilage. here, genes encoding four wnt ligands are up-regulated in muscle-less embryos and in the case of wnt we show specific up-regulation in the elbow and shoulder joint region. two of the up-regulated genes have not previously been associated with skeletal development. the wnt signalling antagonist sfrp is also up-regulated specifically in the joint region. the majority of known wnt target genes affected are down-regulated, perhaps due to increased expression of negative regulators sfrp and dkk and down-regulation of the fzd receptor; however ligands and agonists rspo and rspo are up-regulated as are some target genes indicating effects at multiple levels of regulation of the pathway. it is interesting that a number of the up-regulated targets feedback as negative regulators of the pathway. it is now important to functionally test the mechanisms linking mechanical stimulation with wnt signalling. this work provides sets of candidate genes to use in functional assays to excavate this important link. understanding how mechanical stimuli influence the wnt signalling pathway would be a major step forward in understanding how mechanical cues work together with classical molecular positional information to guide spatially appropriate tissue differentiation and provide indications of how conditions can be effectively recreated in vitro to guide stem cell differentiation. in situ hybridisation analysis showed altered gene expression of wnt pathway genes in the developing shoulder and elbow joints; one down regulated and two upregulated. changes in the spatial restriction of sfrp and wnt expression were also seen. this does not represent a delay in normal expression in the mutant because the altered pattern is not reminiscent of earlier stages and the changes are consistent with altered patterning of the tissue territories in the forming joint and the fusion phenotype seen in the mutant, with cartilage forming across the joint at later stages. in this study differential expression in developing skeletal rudiments is documented in the absence of limb muscle; this will include genes that respond to lack of mechanical stimulation but perhaps also as a paracrine response to adjacent muscle cells. we know that phenotypic effects on ossification and joint formation are due to the lack of mechanical stimulation rather than physical absence of muscle cells because phenotypic analysis of a range of mouse mutants where muscle is immobile, reduced or absent have similar effects and we see similar effects in immobilised chick embryos. therefore, although some of the genes identified here may respond to lack of adjacent muscle tissue, many must underlie the phenotypic effects seen in response to lack of mechanical stimulation. this is further supported by the overlap of some of the genes identified here and in skeletal cells in culture or adult tissues, in response to mechanical stimulation. another important limitation in this work is the possibility that a proportion of the down-regulated genes may be due to contamination of the dissected control humeri with adjacent mesenchyme/muscle, since this is being compared to tissue from muscle-less embryos. although care was taken with the dissections, it is impossible to be sure that all muscle tissue was eliminated from the control. the down-regulated gene set also showed enrichment for muscle associated genes, consistent with possible contamination of the dissected control humeri by neighbouring muscle. to inform this we also sequenced the transcriptome of mesenchyme adjacent to the humerus of control embryos at ts and compared it to the transcriptome of control humeri. we then cross referenced this to the down-regulated gene set in control versus muscle-less humeri, noting any genes enriched more than fold in mesenchyme compared to control humeri; these are indicated in column of additional file 1: table s it is possible that these genes are involved in both cartilage and muscle development so no genes have been removed from the data set, however, de genes also showing higher expression in mesenchyme compared to control humeri must be treated with caution with respect to a skeletal specific response to mechanical stimulation. such genes have not been prioritised in any of our subsequent exploration of candidate mechanosensitive genes. the developing humerus at ts constitutes different cell and tissue populations at different stages of differentiation including the joint region, the perichondrium and the organised zones within the cartilage rudiment. therefore the experimental design employed here will capture genes associated with different cells types at different stages of differentiation. it will now be important to sort out which cells and tissues have altered expression of specific genes. this can be addressed for a sub set of genes by in situ hybridisation, with an initial analysis of genes presented in figure  it can be addressed in a high throughput manner by isolating specific cell populations using laser microdissection from tissue sections, purification of rna and quantitative rt-pcr gene expression profiling, comparing control and mutant tissue from,, for example the hypertrophic, prehypertrophic or the elbow joint region alone. we used both rna-sequencing and microarray technologies in parallel to determine differential expression. microarray technology has been utilised to determine expression of chondrogenic and osteogenic genes from developing whole tissues, and from in vitro differentiation procedures. the use of rna-seq technology to describe the transcriptome is more recent. previous direct comparisons between microarray and rna sequencing-based approaches to reveal alterations in gene expression between tissues reported that rna-seq identified more de genes. we also found that rna-seq is more sensitive in reproducibly detecting alterations in gene expression, detecting more genes altered at lower quantitative levels. this was further emphasised by reducing the stringency of the statistical analysis to p ≤ , which increased the number of genes detected by microarray specifically. an example of the importance of the increased sensitivity and reproducibility of rna-seq is shown by the spp gene which did not show statistical significance by microarray but has been verified by qrt-pcr and in situ hybridisation. the larger dynamic range and higher reproducibility across replicates has also been found in other studies.  this study examines the set of genes active at a key stage of skeletal development and reveals the genes that are differentially regulated in the developing humerus when skeletal muscle is absent. since we previously showed that the lack of muscle contractions leads to common phenotypic defects in both ossification and joint formation in several chick and mouse models, this provides an insight into the genome wide alterations in gene transcription that take place when the mechanical environment is altered. given the importance of appropriate mechanical stimulation generated by embryo movement on skeletal development we postulated that mechanical stimuli must integrate with biochemical cell signalling pathways known to be essential for normal development. we show that multiple signalling pathways are affected, with components of the wnt signalling pathway most strongly disturbed including wnt ligands and both down-regulation and up-regulation of target genes. down-regulated genes include cd dll and fgf which are involved in further cellular interactions during joint formation or feed into other important cell communication events. among the up-regulated wnt targets are several genes that feed back into the wnt pathway itself as antagonists or agonists. this finding, together with alteration of cytoskeletal components, indicates the biological processes involved in integrating biophysical stimuli during cell differentiation and patterning. understanding the mechanistic basis for how developing cells interpret and respond to biophysical cues is a major challenge, relevant to all developing systems, and will impact our ability to control differentiation of progenitor cells for regenerative therapies. this work is an early step in unravelling the mechanistic basis of biophysical regulation of skeletal development and provides a focus for future studies. 
this work identifies key developmental regulatory genes impacted by altered mechanical stimulation, sheds light on the molecular mechanisms that interpret mechanical stimulation during skeletal development and provides valuable resources for further investigation of the mechanistic basis of mechanoregulation. we use a mutant mouse model with altered mechanical stimulation due to the absence of limb skeletal muscle where muscle-less embryos show specific defects in skeletal elements including delayed ossification, changes in the size and shape of cartilage rudiments and joint fusion. spatial gene expression analysis showed that both a wnt ligand encoding gene and a pathway antagonist are up-regulated specifically in the developing joint line, while the expression of a wnt target gene, cd is no longer detectable in muscle-less embryos. mechanical stimulation is necessary for regulating correct formation of the skeleton. here we test the hypothesis that mechanical stimulation of the embryonic skeletal system impacts expression levels of genes implicated in developmentally important signalling pathways in a genome wide approach. we analysed the resulting differentially expressed gene sets using gene ontology annotations to identify significant enrichment of genes associated with particular biological processes, showing that removal of mechanical stimuli from muscle contractions affected genes associated with development and differentiation, cytoskeletal architecture and cell signalling. the identification of genes associated with the cytoskeleton that are down-regulated in the absence of muscle indicates a number of candidate genes that are both mechanoresponsive and potentially involved in mechanotransduction, converting a mechanical stimulus into a transcriptional response. we found that independent genes were down-regulated and genes up-regulated in humeri from muscle-less spd embryos compared to littermate controls. in particular it highlights the wnt signalling pathway as a potential point of integration of mechanical and molecular signalling and cytoskeletal components as mediators of the response.
 mechanical stimulation is necessary for regulating correct formation of the skeleton. here we test the hypothesis that mechanical stimulation of the embryonic skeletal system impacts expression levels of genes implicated in developmentally important signalling pathways in a genome wide approach. we use a mutant mouse model with altered mechanical stimulation due to the absence of limb skeletal muscle where muscle-less embryos show specific defects in skeletal elements including delayed ossification, changes in the size and shape of cartilage rudiments and joint fusion. we used microarray and rna sequencing analysis tools to identify differentially expressed genes between muscle-less and control embryonic humerus tissue.  we found that independent genes were down-regulated and genes up-regulated in humeri from muscle-less spd embryos compared to littermate controls. we analysed the resulting differentially expressed gene sets using gene ontology annotations to identify significant enrichment of genes associated with particular biological processes, showing that removal of mechanical stimuli from muscle contractions affected genes associated with development and differentiation, cytoskeletal architecture and cell signalling. among cell signalling pathways, the most strongly disturbed was wnt signalling, with genes including pathway target genes affected. spatial gene expression analysis showed that both a wnt ligand encoding gene and a pathway antagonist are up-regulated specifically in the developing joint line, while the expression of a wnt target gene, cd is no longer detectable in muscle-less embryos. the identification of genes associated with the cytoskeleton that are down-regulated in the absence of muscle indicates a number of candidate genes that are both mechanoresponsive and potentially involved in mechanotransduction, converting a mechanical stimulus into a transcriptional response.  this work identifies key developmental regulatory genes impacted by altered mechanical stimulation, sheds light on the molecular mechanisms that interpret mechanical stimulation during skeletal development and provides valuable resources for further investigation of the mechanistic basis of mechanoregulation. in particular it highlights the wnt signalling pathway as a potential point of integration of mechanical and molecular signalling and cytoskeletal components as mediators of the response. skeletal developmentmechanical stimulationtranscriptomemechanosensitive geneswnt signallingspdcytoskeleton 