BACKGROUND
the accumulation of genome-wide sequence data for model organisms is of obvious benefit for many kinds of scientific analysis.
although the sheep is of great economic importance and has been used as a model organism to study such diverse topics as fracture healing  <cit> , ovarian function  <cit> , hypernatremic hypertension  <cit> , and pulmonary function  <cit> , relatively little sequence information is available for this organism, and dna sequences are available for only a small number of sheep genes.
<dig> loci,  <dig> of which are designated as genes, are currently listed in the arkdb sheep database  <cit> .
in unigene  <cit>  build  <dig> for ovis aries, a total of  <dig>  est and mrna sequences form a total of  <dig> clusters, and the ebml database  <cit>  currently contains  <dig> ests and  <dig> mrna entries for sheep.
fractures, and especially osteoporotic fractures in the elderly, represent a major public health problem.
as populations age, the costs of treating osteoporotic fractures, currently billions of dollars a year in the united states alone, are predicted to dramatically increase  <cit> .
consequently, a better understanding of the genetic processes underlying fracture healing is of great medical relevance.
the process of fracture healing is a regenerative process that in many ways is similar to bone development.
the fracture repair process goes through many stages, beginning with an inflammatory response and the recruitment and proliferation of undifferentiated mesenchymal cells.
mesenchymal cells from the periosteum near the fracture site differentiate into osteoblasts and induce intramembranous ossification resulting in the formation of new bone.
simultaneously, cartilage is formed at the fracture site and undergoes enchondral ossification.
the newly produced bone then undergoes extensive remodeling until the original shape and structure of the bone is restored.
many of the molecular signals and processes involved in fracture healing closely resemble those involved in bone development  <cit> .
in the present work, we have extracted  <dig> thousand ests from libraries developed from healing bone in a sheep model of fracture healing.
we clustered our sequences together with previously identified ests, and identified  <dig> contigs, the majority of which represented previously unsequenced sheep genes.
these newly identified sequences were used to perform rt-pcr on  <dig> genes from the postfracture day  <dig>   <dig>   <dig>  and  <dig> libraries.
we confirmed differential expression for genes previously known to be involved in fracture healing and demonstrated differential expression for numerous genes not previously known to be involved in fracture healing.
RESULTS
ests and assembly into contigs and creation of an ovis aries sequence database
in addition to the  <dig> sheep ests generated from three libraries in the current project , we extracted  <dig> ests and  <dig> mrna entries from the embl database .
<dig> sequences were rejected because less than  <dig> bp remained unmasked following quality control procedures.
the lengths of the ests ranged from  <dig> to  <dig> with an average length of  <dig> nucleotides .
all bases beyond  <dig> bp were masked.
using our sequences as well as the previously available sequences,  <dig> contigs were assembled with an average length of  <dig> nucleotides .
<dig> contigs were shorter than  <dig> nucleotides and were excluded from further analysis.
on average, each contig was assembled from  <dig>  ests from all libraries, with a range of  <dig> to  <dig> ests per contig .
if the analysis is restricted to ests from the libraries described in this work, then each contig contained an average of  <dig>  ests .
the contigs comprised of the largest number of ests were identified as collagen iα <dig> , collagen iα <dig> , and osteonectin .
a total of  <dig> contigs contained only a single est clone.
we used the genenest package  <cit>  to analyze all available mrna and est sequences and to present the sequences in a user-friendly internet site.
the main features of the genenest sheep database are summarized in fig.
<dig>
blast analysis of ovis aries contigs to assign putative gene identities based on homology
blast searches were used to attempt to assign putative identities to the contig sequences identified in this study.
analysis against the ncbi refseq protein sequence database was performed, and  <dig> contigs demonstrated a best e-value of  <dig>  or lower .
we used an arbitrary e-value cutoff of 10- <dig> to identify significant homology to a known gene and allow a probable assignment of a sheep contig.
with this cutoff, a total of  <dig> contigs demonstrated significant homology to at least one other protein in the refseq protein database and could be assigned a putative identity, consisting of a total of  <dig> distinct proteins.
in cases where hits to a proteins from several species were identified, the most significant match was used to assign a putative identity.
<dig> contigs blasted to an ovis aries refseq.
a total of  <dig> contigs  had e-values of less than 10- <dig> and are thus likely to represent true orthologs.
of the  <dig> contigs,  <dig> were assembled from ests only from the current project.
<dig> were assigned to  <dig> distinct refseq sequences by virtue of blastx e-values of 10- <dig> or less.
these sequences represent previously unidentified sheep genes.
the sequences for these genes are available in fasta format with the online supplemental material as
gene-ontology analysis of the sheep fracture libraries
go analysis was performed by assigning annotations to the sheep contigs from the mgi annotations of putative murine homologs  in order to get an overview of the subset of genes expressed in healing fracture tissue of the sheep.
fig.
3b provides an overview of the results of this blast analysis.
putative homologs were found for  <dig> distinct sheep genes, some of which were defined from multiple contig sequences.
mgi annotations were available for  <dig> of the  <dig> genes.
the results of the go analysis using these annotations are summarized in fig.
<dig>  many of the most frequently annotated terms in the subontology biological process, such as development, signal transduction, transcription, biosynthesis, and phosphate metabolism, reflect processes known to be involved in fracture healing and bone development.
the distribution of annotations to terms in the subontology cellular component shows that the gene products corresponding to the ests identified in this work are active in many different cellular and extracellular locations.
a comparison of the sheep fracture libraries with normal human bone libraries
during development and in related processes such as fracture healing, genes exhibit complex patterns of spatial and temporal regulation.
the identification of genes that are differentially expressed during fracture healing therefore represents an important initial investigation to discover genes that are essential for fracture healing.
in this work, we generated two est libraries from early fracture healing stages  with the goal of identifying genes important in the earliest stages of fracture healing.
following fracture, initial hemorrhage into the fractured bone is followed by hemostasis and a phase of inflammation, which lasts up to two weeks in the sheep .
intramembranous ossification begins around day  <dig>  therefore, we reasoned that genes that are overexpressed in the pf <dig> and pf <dig> libraries could represent genes with potential importance for the initiation of new bone formation.
to estimate gene expression levels in the two libraries, we compared est sequence counts using a two-tailed fisher exact test.
this analysis revealed statistically significant differential expression for only three genes; tartrate resistant acid phosphatase  <dig>  increased by a fold change of  <dig>  , cytochrome c oxidase subunit i  increased by a fold change of  <dig>  , and perlecan decreased by a fold change of  <dig>  .
we did not perform such a comparison using the third est library, because the latter was derived from a mix of time points using pcr amplification of cdna inserts in order to get a broader overview of the genes involved in the many stages of fracture healing in sheep.
rna samples from intact sheep bone were not available.
therefore, we chose to compare the est distribution in the pf <dig> and pf <dig> libraries to that of two resting human bone est libraries.
we reasoned that genes that were significantly more highly expressed in the sheep libraries could represent candidate fracture-healing related genes that might warrant closer investigation.
a total of  <dig> genes with benjamini-hochberg-corrected p-values less than an arbitrary threshold of  <dig>  were identified as putatively differentially expressed.
table  <dig> gives details on the genes and number of ests found .
links to corresponding contigs in the genenest database are given in .
it is striking that  <dig> of the  <dig> genes  had previously been known to be involved with developmental processes in some way .
eleven of the  <dig> genes were previously known to be involved in bone development or fracture healing.
given that fracture healing is thought to recapitulate bone development on a molecular level  <cit> , these findings are suggestive that our est analysis captured some essential aspects of fracture healing.
expression analysis of candidate fracture-healing related genes
we then reasoned that differential expression during the course of fracture healing could be another kind of indicator for genes with potential importance in fracture healing.
therefore, we performed rt-pcr expression analysis using samples from postfracture day  <dig>   <dig>   <dig>  and  <dig>  the rna source for days  <dig> and  <dig> was identical to that used for the pf <dig> and pf <dig> est libraries, and the source for day  <dig> was one of the rna samples used to construct the pooled est library.
the day  <dig> rna sample was not used for any of the est libraries.
samples from normal sheep bone were not available.
pcr primers were designed for  <dig> of the genes in table  <dig> and for an additional  <dig> candidate genes , that were chosen for biological interest or because of tendencies toward overexpression that did not reach statistical significance.
almost all genes showed a large drop in expression at the 42-day time point.
early stage callus tissue is a metabolically highly active tissue.
in comparison, healthy bone is less metabolically active, which is not surprising given that cells make up only around 2% of the total mass of bone.
it has previously been noted that the expression of many genes related to fracture healing drops to very low or undetectable levels at  <dig> weeks after fracture in a rat model  <cit> .
in our experiments,  <dig> of the  <dig> genes  tested by anova over all four timepoints were found to be significantly differentially expressed, a proportion that dropped to 96% if only the first three time points were considered, and 31% for a comparison between only the first two time points.
presents the normalized expression values determined in these experiments.
we then performed hierarchical clustering to produce a graphic representation of the the expression patterns for the genes investigated in rt-pcr.
we used the gap-stat method  <cit>  to estimate the number of clusters in the data .
the estimated number of clusters is two.
however, the gap function rises again with peaks at  <dig> and  <dig> clusters, suggesting that there are less well defined groups of four and of six clusters in the data.
for the purposes of discussion and visualization, we chose to partition the genes into six clusters .
clusters a, b, e and f demonstrated high frequencies of genes of specific functional classes .
discussion
in the present work, we have performed large-scale est sequencing and analysis in order to generate sufficient sequence information to study fracture healing at a genomic level in the sheep for the first time.
the results of our study are of interest not only in the context of fracture healing and bone development, but will also provide useful information for all fields of research that use the sheep as a model organism.
the ests generated in the present work represent an increase in the number of available ests by a factor of over  <dig> , and provide at least partial sequences for over  <dig> sheep genes never before sequenced.
the sequences can be downloaded from the online supplemental site and are also available in the genenest sheep database.
genomic approaches to the sheep have great potential to accelerate progress in research areas using the sheep as a model organism.
for instance, the first study using an ovine cdna microarray prepared from unpublished est libraries was very recently published.
a number of genes differentially expressed in sheep resistant and susceptible to gastrointestinal nematode infection were identified  <cit> .
although we now have initial concepts of the molecular processes involved in fracture healing, many questions remain to be answered before a comprehensive picture of the genetic programs involved in fracture healing can emerge.
an in-depth understanding of the genes involved in fracture healing and the regulatory mechanisms that control them will be prerequisite for developing optimal treatments for disturbances of fracture healing and will have relevance to other disorders such as osteoporosis.
at present, we still do not have a comprehensive list of key genes and gene products involved in bone development and fracture healing.
in this study, we have performed the first large-scale est sequencing project using a model for fracture healing and have identified numerous differentially regulated genes not previously known to be involved in fracture healing.
est sequencing gives a nearly unbiased view of highly expressed sequences in a tissue , and therefore offers some advantages over candidate gene approaches and even over microarray-based approaches that may not cover the entire genome.
in this work, we have generated a profile of postfracture day  <dig> and day  <dig> genetic expression using est libraries produced by automated plasmid purification.
no normalization procedures were applied to select ests for sequencing.
therefore, we believe that the distribution of ests in these libraries gives a nearly unbiased view of genetic expression at these stages of fracture healing.
in addition, we generated a third est library from a pool of later stages of fracture healing and used rt-pcr to examine differential expression of  <dig> of the  <dig> genes found to be enriched in the pf <dig> and pf <dig> libraries and  <dig> other genes .
previous genomic approaches to fracture healing have involved analysis of a rat femur fracture model based mainly on microarray expression analysis, but different experimental and statistical approaches were used  <cit> .
these studies have collectively identified thousands of genes that are differentially expressed in fracture healing.
the genes were grouped by the authors into a number of functional classes, which overlap to a large extent with the classes identified in the present investigation, including extracellular matrix proteins, ribosomal proteins, resorption/remodeling, transcriptional regulation and signal transduction.
however, the specific genes belonging to the functional classes are often different among the three studies cited above as well as in comparison to our results.
for instance, hadjiargyrou and coworkers  identified  <dig> differentially expressed ribosomal genes; in the present study,  <dig> differentially expressed ribosomal genes were identified.
with the exception of ribosomal protein sa, there is no overlap between the two lists.
this suggests that we are just beginning to identify to the full complement of genes involved in fracture healing.
genes differentially expressed in fracture healing
many of the differentially expressed genes we identified were previously known to be involved in various biological processes involved in bone development or fracture healing , suggesting that our approach has captured some essential aspects of fracture healing.
additionally, we have demonstrated differential expression for many genes not previously known to be involved in fracture healing.
the differentially expressed genes can be divided into several different classes, as will be discussed below.
three of these classes, extracellular matrix proteins, resorption/remodeling/inflammation, and transcriptional regulation/signal transduction, have been well studied in the context of fracture healing, and our results have identified both previously known and novel differentially regulated genes with these functional roles.
in the following, we describe three other classes with numerous differentially regulated genes, angiogenesis, free-radical control, and ribosomal genes.
these functional classes have received less attention to date, although they are known to be involved in the process of fracture healing.
fracture-related differential expression had not previously been shown for the great majority of genes in these classes .
these observations thus represent starting points for future studies on the role of processes such as angiogenesis and free-radical control in fracture healing.
ribosomal genes
the single largest group of putatively upregulated genes in our analysis was made up of ribosomal proteins.
there are over  <dig> ribosomal proteins  <cit> , which are not necessarily expressed in concert  <cit> .
we found  <dig> genes coding for ribosomal proteins to be significantly overexpressed in our analysis of est distribution compared to normal human bone.
all of these genes, as well as seven additional genes coding for ribosomal proteins  showed differential expression in the rt-pcr analysis, with expression levels peaking at postfracture days  <dig> and  <dig> .
we additionally identified four translation factors as being overexpressed in our est analysis .
rt-pcr for two of these genes confirmed differential expression.
to the best of our knowledge, a specific role in fracture healing has not been identified for all but two of these genes to date, although some of them are known to be differentially expressed in certain developmental processes , and a number of other ribosomal genes are known to be differentially expressed in fracture healing  <cit> .
cluster b  contained  <dig> genes.
interestingly,  <dig> of the  <dig> ribosomal genes analyzed by rt-pcr, and both of the translation factors analyzed by rt-pcr were in this cluster, whose expression levels peaked during the early phases of fracture healing at days  <dig> and  <dig> postfracture, suggesting that there is an especially high level of protein synthesis at these stages.
extracellular matrix proteins
several matrix proteins were found to be differentially regulated.
the gene with the highest overall expression, collagen iα <dig>  is the main protein component of bone, and the fact that it is one of the most highly upregulated genes is not surprising.
two other collagens and osteonectin were also found to be upregulated by est analysis .
we investigated these genes and four other collagen genes and two proteoglycans by rt-pcr and showed differential expression for all genes.
in general, the expression of these matrix genes was highest at postfracture day  <dig> or  <dig> but was still relatively high at day  <dig>  ibsp is an osteoblast marker that is a major noncollagenous structural protein of the bone matrix.
ibsp showed an expression pattern different from that of the other matrix genes, with significant expression beginning at day  <dig>  peaking at day  <dig>  and continuing until day  <dig>
the lysyl-trna synthetase kars has no known involvement in fracture healing.
given the important role of lysine crosslinks in the biosynthesis of collagen, it is interesting that lysyl-trna synthetase was the only trna synthetase gene that was overexpressed in our data.
serpinh <dig> was also found to be significantly overexpressed in the pf <dig> and pf <dig> ests.
it is a molecular chaperone involved in the maturation of collagen molecules  <cit> .
this is presumably important for bone development because serpinh <dig> is localized to regions of type i collagen production in developing murine femurs and tibiae  <cit> .
angiogenesis-related genes
it has long been known that vascular invasion is necessary for bone differentiation  <cit> , and angiogenesis is a key process for fracture healing  <cit> .
a number of genes related to angiogenesis have previously been shown to be upregulated during bone repair, such as vascular endothelial growth factor  <cit> .
col3a <dig> and col4a <dig> were shown to be overexpressed in the pf <dig> and pf <dig> ests .
both of these collagens have known roles in angiogenesis  <cit> .
six of the eight genes in our dataset with prominent roles in angiogenesis  were in cluster a. the smooth muscle marker csrp <dig>  <cit>  and the tgfβ-family auxiliary receptor endoglin are required for angiogenesis and heart development  <cit> .
hdgf is a highly expressed vascular endothelial cell protein  <cit> .
the genes of this cluster show especially strong expression on day  <dig> postfracture with lesser amounts of expression on days  <dig> and  <dig>  which is not surprising given that new blood vessel formation is a prerequisite for the formation of new bone from callus tissue.
the other two genes, col3a <dig> and tagln <dig>  were in cluster b, which like cluster a is characterized by high early expression , but shows a stronger drop in expression at the  <dig> day timepoint.
resorption, remodeling and inflammation
several genes that probably play a role in the inflammatory response characteristic of early stages of fracture healing  were found to be overexpressed in the pf <dig> and pf <dig> ests .
resorption of cartilage and bone mediated by osteoclasts is an important part of the remodeling processes involved in new bone formation in fracture healing.
the main enzymes involved are thought to be the cathepsins and the matrix metalloproteinases , which act in a concerted fashion.
although not all details are clear, our current understanding of this process suggests that osteoclasts create an acidic environment in their resorption lacunae, which results in dissolution of the mineral, and then secrete cysteine proteinases that are active at a low ph, to initiate proteolysis of the proteinaceous bone matrix.
finally, mmps exert their activity, once the ph has increased sufficiently  <cit> .
our data has demonstrated differential expression for several enzymes from both classes.
interestingly, four of the five genes in rt-pcr cluster e encode enzymes with roles in osteogenesis .
the first three genes are expressed by osteoclasts.
the expression of genes in this cluster was minimal at day  <dig> but peaked at day  <dig> postfracture, suggesting that these enzymes might be particularly important for the processes involved in the bone formation that becomes visible by day  <dig>
we showed differential expression for mmp <dig>  mmp <dig>  mmp <dig>  mmp <dig>  and mmp <dig>  all of these mmps had previously known roles in bone development except mmp <dig>  providing further evidence for the similarity of bone development and fracture healing.
mmp <dig>  mmp <dig>  and mmp <dig> are differentially regulated in scarless fetal wound healing  <cit>  and during osteogenesis  <cit> , which is interesting because bone is one of the few adult tissues that can heal without scarring.
mmp <dig> previously had no known role in skeletal development or fracture healing, but it is highly expressed in dermal wounds, suggesting a role in wound repair  <cit> .
the highest expression of mmp <dig> was observed in the day  <dig> and  <dig> stages.
cluster f contained only two genes, cystatin c and tpm <dig>  these genes were the only ones with an expression profile that peaked at the last time point .
cystatin c  can inhibit bone resorption and osteoclast formation  <cit> .
osteoclast activity begins early and increases over the course of fracture healing  <cit> , and one may speculate that cst <dig> may be important for regulation of osteoclasts at this time point.
control of free radicals
vascular invasion of ossified cartilage during enchondral ossification is associated with breakdown of tissue by the release of lytic enzymes by invading cells such as macrophages and endothelial cells.
this process is likely to involve the production of reactive oxygen species   <cit> .
at present, little is known about the mechanisms by which unwanted tissue damage by ros is controlled during fracture healing.
the glutathione peroxidases  are enzymes that catalyze the reduction of hydrogen peroxide, organic hydroperoxide, and lipid peroxides by reduced glutathione.
they are thus involved in the protection of cells against oxidative damage.
although none of the four gpx genes we tested in rt-pcr were individually statistically significantly overexpressed in the est analysis, there were no ests for any of these gpx genes in normal bone and a total of  <dig> ests for the four gpx genes in the pf <dig> and pf <dig> est libraries.
we showed significant differential expression patterns for each of these genes by rt-pcr, although they did not show a uniform expression pattern .
hmox <dig> was also found to be significantly overexpressed in the pf <dig> and pf <dig> est libraries, as has been previously described for postfracture day  <dig> in a rat femur fracture model  <cit> .
hmox <dig> is an inducible heme oxygenase that could conceivably be involved in heme catabolism during resorption of the initial fracture hematoma, although other roles in development have been postulated for this gene  <cit> .
transcription regulation and signal transduction
two membrane-bound proteins with roles in signal transduction were found to be significantly overexpressed in the pf <dig> and pf <dig> libraries.
itm2c is highly expressed during chondro-osteogenic differentiation  <cit> , but its specific function and its role in fracture healing remain unknown.
the g protein gnai <dig> was also found to be overexpressed, but no clues are available as to its particular role in fracture healing.
signal transduction and modification of transcriptional programs by transcription factors represent important phenomena in development, and presumably are essential for fracture healing.
nevertheless, the est counts for the transcription factors examined in this study were too low to reach statistical significance, even though in many cases ests for transcription factors were found only in the pf <dig> and pf <dig> libraries but not in the human normal bone libraries.
for instance, we identified only one est each for the transcription factor runx- <dig> and osterix, which are both key players in skeletogenesis  <cit> .
we performed rt-pcr analysis on a number of transcription factors with relatively large numbers of ests in our libraries .
we were able to demonstrate differential expression with the highest expression at days  <dig> and  <dig>  for junb, which among other roles is a positive regulator controlling primarily osteoblast as well as osteoclast activity  <cit> .
morf4l <dig>  which may play a role in mechanotransduction  <cit> , and tcf <dig>  which may mediate wnt signaling during limb development  <cit>  also showed highest expression levels in the first two time points.
additionally, we showed differential expression for nsep <dig>  ppib, and rara, for which to the best of our knowledge no previous information about differential expression during bone development or fracture healing was available.
CONCLUSIONS
in this work we present approximately  <dig>  ests from the domestic sheep.
this represents an important resource to researchers interested in the sheep as a model organism and greatly expands the number of gene sequences available for ovis aries .the sequences are freely available in genenest and the public sequence databases.
we have used the sequences we generated as a tool to investigate the early stages of fracture healing and have identified numerous genes that are differentially regulated in early callus tissues, many of which were not previously known to have any specific relation to fracture healing or bone development.
these genes will be valuable leads for further research into the molecular mechanisms of fracture healing.

 the accumulation of genome-wide sequence data for model organisms is of obvious benefit for many kinds of scientific analysis. although the sheep is of great economic importance and has been used as a model organism to study such diverse topics as fracture healing, ovarian function, hypernatremic hypertension, and pulmonary function, relatively little sequence information is available for this organism, and dna sequences are available for only a small number of sheep genes. loci, of which are designated as genes, are currently listed in the arkdb sheep database. in unigene build for ovis aries, a total of est and mrna sequences form a total of clusters, and the ebml database currently contains ests and mrna entries for sheep. fractures, and especially osteoporotic fractures in the elderly, represent a major public health problem. as populations age, the costs of treating osteoporotic fractures, currently billions of dollars a year in the united states alone, are predicted to dramatically increase. consequently, a better understanding of the genetic processes underlying fracture healing is of great medical relevance. the process of fracture healing is a regenerative process that in many ways is similar to bone development. the fracture repair process goes through many stages, beginning with an inflammatory response and the recruitment and proliferation of undifferentiated mesenchymal cells. mesenchymal cells from the periosteum near the fracture site differentiate into osteoblasts and induce intramembranous ossification resulting in the formation of new bone. simultaneously, cartilage is formed at the fracture site and undergoes enchondral ossification. the newly produced bone then undergoes extensive remodeling until the original shape and structure of the bone is restored. many of the molecular signals and processes involved in fracture healing closely resemble those involved in bone development. in the present work, we have extracted thousand ests from libraries developed from healing bone in a sheep model of fracture healing. we clustered our sequences together with previously identified ests, and identified contigs, the majority of which represented previously unsequenced sheep genes. these newly identified sequences were used to perform rt-pcr on genes from the postfracture day and libraries. we confirmed differential expression for genes previously known to be involved in fracture healing and demonstrated differential expression for numerous genes not previously known to be involved in fracture healing.  ests and assembly into contigs and creation of an ovis aries sequence database in addition to the sheep ests generated from three libraries in the current project, we extracted ests and mrna entries from the embl database. sequences were rejected because less than bp remained unmasked following quality control procedures. the lengths of the ests ranged from to with an average length of nucleotides. all bases beyond bp were masked. using our sequences as well as the previously available sequences, contigs were assembled with an average length of nucleotides. contigs were shorter than nucleotides and were excluded from further analysis. on average, each contig was assembled from ests from all libraries, with a range of to ests per contig. if the analysis is restricted to ests from the libraries described in this work, then each contig contained an average of ests. the contigs comprised of the largest number of ests were identified as collagen iα, collagen iα, and osteonectin. a total of contigs contained only a single est clone. we used the genenest package to analyze all available mrna and est sequences and to present the sequences in a user-friendly internet site. the main features of the genenest sheep database are summarized in fig. blast analysis of ovis aries contigs to assign putative gene identities based on homology blast searches were used to attempt to assign putative identities to the contig sequences identified in this study. analysis against the ncbi refseq protein sequence database was performed, and contigs demonstrated a best e-value of or lower. we used an arbitrary e-value cutoff of 10- to identify significant homology to a known gene and allow a probable assignment of a sheep contig. with this cutoff, a total of contigs demonstrated significant homology to at least one other protein in the refseq protein database and could be assigned a putative identity, consisting of a total of distinct proteins. in cases where hits to a proteins from several species were identified, the most significant match was used to assign a putative identity. contigs blasted to an ovis aries refseq. a total of contigs had e-values of less than 10- and are thus likely to represent true orthologs. of the contigs, were assembled from ests only from the current project. were assigned to distinct refseq sequences by virtue of blastx e-values of 10- or less. these sequences represent previously unidentified sheep genes. the sequences for these genes are available in fasta format with the online supplemental material as gene-ontology analysis of the sheep fracture libraries go analysis was performed by assigning annotations to the sheep contigs from the mgi annotations of putative murine homologs in order to get an overview of the subset of genes expressed in healing fracture tissue of the sheep. fig. 3b provides an overview of the results of this blast analysis. putative homologs were found for distinct sheep genes, some of which were defined from multiple contig sequences. mgi annotations were available for of the genes. the results of the go analysis using these annotations are summarized in fig. many of the most frequently annotated terms in the subontology biological process, such as development, signal transduction, transcription, biosynthesis, and phosphate metabolism, reflect processes known to be involved in fracture healing and bone development. the distribution of annotations to terms in the subontology cellular component shows that the gene products corresponding to the ests identified in this work are active in many different cellular and extracellular locations. a comparison of the sheep fracture libraries with normal human bone libraries during development and in related processes such as fracture healing, genes exhibit complex patterns of spatial and temporal regulation. the identification of genes that are differentially expressed during fracture healing therefore represents an important initial investigation to discover genes that are essential for fracture healing. in this work, we generated two est libraries from early fracture healing stages with the goal of identifying genes important in the earliest stages of fracture healing. following fracture, initial hemorrhage into the fractured bone is followed by hemostasis and a phase of inflammation, which lasts up to two weeks in the sheep. intramembranous ossification begins around day therefore, we reasoned that genes that are overexpressed in the pf and pf libraries could represent genes with potential importance for the initiation of new bone formation. to estimate gene expression levels in the two libraries, we compared est sequence counts using a two-tailed fisher exact test. this analysis revealed statistically significant differential expression for only three genes; tartrate resistant acid phosphatase increased by a fold change of, cytochrome c oxidase subunit i increased by a fold change of, and perlecan decreased by a fold change of. we did not perform such a comparison using the third est library, because the latter was derived from a mix of time points using pcr amplification of cdna inserts in order to get a broader overview of the genes involved in the many stages of fracture healing in sheep. rna samples from intact sheep bone were not available. therefore, we chose to compare the est distribution in the pf and pf libraries to that of two resting human bone est libraries. we reasoned that genes that were significantly more highly expressed in the sheep libraries could represent candidate fracture-healing related genes that might warrant closer investigation. a total of genes with benjamini-hochberg-corrected p-values less than an arbitrary threshold of were identified as putatively differentially expressed. table gives details on the genes and number of ests found. links to corresponding contigs in the genenest database are given in. it is striking that of the genes had previously been known to be involved with developmental processes in some way. eleven of the genes were previously known to be involved in bone development or fracture healing. given that fracture healing is thought to recapitulate bone development on a molecular level, these findings are suggestive that our est analysis captured some essential aspects of fracture healing. expression analysis of candidate fracture-healing related genes we then reasoned that differential expression during the course of fracture healing could be another kind of indicator for genes with potential importance in fracture healing. therefore, we performed rt-pcr expression analysis using samples from postfracture day and the rna source for days and was identical to that used for the pf and pf est libraries, and the source for day was one of the rna samples used to construct the pooled est library. the day rna sample was not used for any of the est libraries. samples from normal sheep bone were not available. pcr primers were designed for of the genes in table and for an additional candidate genes, that were chosen for biological interest or because of tendencies toward overexpression that did not reach statistical significance. almost all genes showed a large drop in expression at the 42-day time point. early stage callus tissue is a metabolically highly active tissue. in comparison, healthy bone is less metabolically active, which is not surprising given that cells make up only around 2% of the total mass of bone. it has previously been noted that the expression of many genes related to fracture healing drops to very low or undetectable levels at weeks after fracture in a rat model. in our experiments, of the genes tested by anova over all four timepoints were found to be significantly differentially expressed, a proportion that dropped to 96% if only the first three time points were considered, and 31% for a comparison between only the first two time points. presents the normalized expression values determined in these experiments. we then performed hierarchical clustering to produce a graphic representation of the the expression patterns for the genes investigated in rt-pcr. we used the gap-stat method to estimate the number of clusters in the data. the estimated number of clusters is two. however, the gap function rises again with peaks at and clusters, suggesting that there are less well defined groups of four and of six clusters in the data. for the purposes of discussion and visualization, we chose to partition the genes into six clusters. clusters a, b, e and f demonstrated high frequencies of genes of specific functional classes. discussion in the present work, we have performed large-scale est sequencing and analysis in order to generate sufficient sequence information to study fracture healing at a genomic level in the sheep for the first time. the results of our study are of interest not only in the context of fracture healing and bone development, but will also provide useful information for all fields of research that use the sheep as a model organism. the ests generated in the present work represent an increase in the number of available ests by a factor of over, and provide at least partial sequences for over sheep genes never before sequenced. the sequences can be downloaded from the online supplemental site and are also available in the genenest sheep database. genomic approaches to the sheep have great potential to accelerate progress in research areas using the sheep as a model organism. for instance, the first study using an ovine cdna microarray prepared from unpublished est libraries was very recently published. a number of genes differentially expressed in sheep resistant and susceptible to gastrointestinal nematode infection were identified. although we now have initial concepts of the molecular processes involved in fracture healing, many questions remain to be answered before a comprehensive picture of the genetic programs involved in fracture healing can emerge. an in-depth understanding of the genes involved in fracture healing and the regulatory mechanisms that control them will be prerequisite for developing optimal treatments for disturbances of fracture healing and will have relevance to other disorders such as osteoporosis. at present, we still do not have a comprehensive list of key genes and gene products involved in bone development and fracture healing. in this study, we have performed the first large-scale est sequencing project using a model for fracture healing and have identified numerous differentially regulated genes not previously known to be involved in fracture healing. est sequencing gives a nearly unbiased view of highly expressed sequences in a tissue, and therefore offers some advantages over candidate gene approaches and even over microarray-based approaches that may not cover the entire genome. in this work, we have generated a profile of postfracture day and day genetic expression using est libraries produced by automated plasmid purification. no normalization procedures were applied to select ests for sequencing. therefore, we believe that the distribution of ests in these libraries gives a nearly unbiased view of genetic expression at these stages of fracture healing. in addition, we generated a third est library from a pool of later stages of fracture healing and used rt-pcr to examine differential expression of of the genes found to be enriched in the pf and pf libraries and other genes. previous genomic approaches to fracture healing have involved analysis of a rat femur fracture model based mainly on microarray expression analysis, but different experimental and statistical approaches were used. these studies have collectively identified thousands of genes that are differentially expressed in fracture healing. the genes were grouped by the authors into a number of functional classes, which overlap to a large extent with the classes identified in the present investigation, including extracellular matrix proteins, ribosomal proteins, resorption/remodeling, transcriptional regulation and signal transduction. however, the specific genes belonging to the functional classes are often different among the three studies cited above as well as in comparison to our results. for instance, hadjiargyrou and coworkers identified differentially expressed ribosomal genes; in the present study, differentially expressed ribosomal genes were identified. with the exception of ribosomal protein sa, there is no overlap between the two lists. this suggests that we are just beginning to identify to the full complement of genes involved in fracture healing. genes differentially expressed in fracture healing many of the differentially expressed genes we identified were previously known to be involved in various biological processes involved in bone development or fracture healing, suggesting that our approach has captured some essential aspects of fracture healing. additionally, we have demonstrated differential expression for many genes not previously known to be involved in fracture healing. the differentially expressed genes can be divided into several different classes, as will be discussed below. three of these classes, extracellular matrix proteins, resorption/remodeling/inflammation, and transcriptional regulation/signal transduction, have been well studied in the context of fracture healing, and our results have identified both previously known and novel differentially regulated genes with these functional roles. in the following, we describe three other classes with numerous differentially regulated genes, angiogenesis, free-radical control, and ribosomal genes. these functional classes have received less attention to date, although they are known to be involved in the process of fracture healing. fracture-related differential expression had not previously been shown for the great majority of genes in these classes. these observations thus represent starting points for future studies on the role of processes such as angiogenesis and free-radical control in fracture healing. ribosomal genes the single largest group of putatively upregulated genes in our analysis was made up of ribosomal proteins. there are over ribosomal proteins, which are not necessarily expressed in concert. we found genes coding for ribosomal proteins to be significantly overexpressed in our analysis of est distribution compared to normal human bone. all of these genes, as well as seven additional genes coding for ribosomal proteins showed differential expression in the rt-pcr analysis, with expression levels peaking at postfracture days and. we additionally identified four translation factors as being overexpressed in our est analysis. rt-pcr for two of these genes confirmed differential expression. to the best of our knowledge, a specific role in fracture healing has not been identified for all but two of these genes to date, although some of them are known to be differentially expressed in certain developmental processes, and a number of other ribosomal genes are known to be differentially expressed in fracture healing. cluster b contained genes. interestingly, of the ribosomal genes analyzed by rt-pcr, and both of the translation factors analyzed by rt-pcr were in this cluster, whose expression levels peaked during the early phases of fracture healing at days and postfracture, suggesting that there is an especially high level of protein synthesis at these stages. extracellular matrix proteins several matrix proteins were found to be differentially regulated. the gene with the highest overall expression, collagen iα is the main protein component of bone, and the fact that it is one of the most highly upregulated genes is not surprising. two other collagens and osteonectin were also found to be upregulated by est analysis. we investigated these genes and four other collagen genes and two proteoglycans by rt-pcr and showed differential expression for all genes. in general, the expression of these matrix genes was highest at postfracture day or but was still relatively high at day ibsp is an osteoblast marker that is a major noncollagenous structural protein of the bone matrix. ibsp showed an expression pattern different from that of the other matrix genes, with significant expression beginning at day peaking at day and continuing until day the lysyl-trna synthetase kars has no known involvement in fracture healing. given the important role of lysine crosslinks in the biosynthesis of collagen, it is interesting that lysyl-trna synthetase was the only trna synthetase gene that was overexpressed in our data. serpinh was also found to be significantly overexpressed in the pf and pf ests. it is a molecular chaperone involved in the maturation of collagen molecules. this is presumably important for bone development because serpinh is localized to regions of type i collagen production in developing murine femurs and tibiae. angiogenesis-related genes it has long been known that vascular invasion is necessary for bone differentiation, and angiogenesis is a key process for fracture healing. a number of genes related to angiogenesis have previously been shown to be upregulated during bone repair, such as vascular endothelial growth factor. col3a and col4a were shown to be overexpressed in the pf and pf ests. both of these collagens have known roles in angiogenesis. six of the eight genes in our dataset with prominent roles in angiogenesis were in cluster a. the smooth muscle marker csrp and the tgfβ-family auxiliary receptor endoglin are required for angiogenesis and heart development. hdgf is a highly expressed vascular endothelial cell protein. the genes of this cluster show especially strong expression on day postfracture with lesser amounts of expression on days and which is not surprising given that new blood vessel formation is a prerequisite for the formation of new bone from callus tissue. the other two genes, col3a and tagln were in cluster b, which like cluster a is characterized by high early expression, but shows a stronger drop in expression at the day timepoint. resorption, remodeling and inflammation several genes that probably play a role in the inflammatory response characteristic of early stages of fracture healing were found to be overexpressed in the pf and pf ests. resorption of cartilage and bone mediated by osteoclasts is an important part of the remodeling processes involved in new bone formation in fracture healing. the main enzymes involved are thought to be the cathepsins and the matrix metalloproteinases, which act in a concerted fashion. although not all details are clear, our current understanding of this process suggests that osteoclasts create an acidic environment in their resorption lacunae, which results in dissolution of the mineral, and then secrete cysteine proteinases that are active at a low ph, to initiate proteolysis of the proteinaceous bone matrix. finally, mmps exert their activity, once the ph has increased sufficiently. our data has demonstrated differential expression for several enzymes from both classes. interestingly, four of the five genes in rt-pcr cluster e encode enzymes with roles in osteogenesis. the first three genes are expressed by osteoclasts. the expression of genes in this cluster was minimal at day but peaked at day postfracture, suggesting that these enzymes might be particularly important for the processes involved in the bone formation that becomes visible by day we showed differential expression for mmp mmp mmp mmp and mmp all of these mmps had previously known roles in bone development except mmp providing further evidence for the similarity of bone development and fracture healing. mmp mmp and mmp are differentially regulated in scarless fetal wound healing and during osteogenesis, which is interesting because bone is one of the few adult tissues that can heal without scarring. mmp previously had no known role in skeletal development or fracture healing, but it is highly expressed in dermal wounds, suggesting a role in wound repair. the highest expression of mmp was observed in the day and stages. cluster f contained only two genes, cystatin c and tpm these genes were the only ones with an expression profile that peaked at the last time point. cystatin c can inhibit bone resorption and osteoclast formation. osteoclast activity begins early and increases over the course of fracture healing, and one may speculate that cst may be important for regulation of osteoclasts at this time point. control of free radicals vascular invasion of ossified cartilage during enchondral ossification is associated with breakdown of tissue by the release of lytic enzymes by invading cells such as macrophages and endothelial cells. this process is likely to involve the production of reactive oxygen species. at present, little is known about the mechanisms by which unwanted tissue damage by ros is controlled during fracture healing. the glutathione peroxidases are enzymes that catalyze the reduction of hydrogen peroxide, organic hydroperoxide, and lipid peroxides by reduced glutathione. they are thus involved in the protection of cells against oxidative damage. although none of the four gpx genes we tested in rt-pcr were individually statistically significantly overexpressed in the est analysis, there were no ests for any of these gpx genes in normal bone and a total of ests for the four gpx genes in the pf and pf est libraries. we showed significant differential expression patterns for each of these genes by rt-pcr, although they did not show a uniform expression pattern. hmox was also found to be significantly overexpressed in the pf and pf est libraries, as has been previously described for postfracture day in a rat femur fracture model. hmox is an inducible heme oxygenase that could conceivably be involved in heme catabolism during resorption of the initial fracture hematoma, although other roles in development have been postulated for this gene. transcription regulation and signal transduction two membrane-bound proteins with roles in signal transduction were found to be significantly overexpressed in the pf and pf libraries. itm2c is highly expressed during chondro-osteogenic differentiation, but its specific function and its role in fracture healing remain unknown. the g protein gnai was also found to be overexpressed, but no clues are available as to its particular role in fracture healing. signal transduction and modification of transcriptional programs by transcription factors represent important phenomena in development, and presumably are essential for fracture healing. nevertheless, the est counts for the transcription factors examined in this study were too low to reach statistical significance, even though in many cases ests for transcription factors were found only in the pf and pf libraries but not in the human normal bone libraries. for instance, we identified only one est each for the transcription factor runx- and osterix, which are both key players in skeletogenesis. we performed rt-pcr analysis on a number of transcription factors with relatively large numbers of ests in our libraries. we were able to demonstrate differential expression with the highest expression at days and for junb, which among other roles is a positive regulator controlling primarily osteoblast as well as osteoclast activity. morf4l which may play a role in mechanotransduction, and tcf which may mediate wnt signaling during limb development also showed highest expression levels in the first two time points. additionally, we showed differential expression for nsep ppib, and rara, for which to the best of our knowledge no previous information about differential expression during bone development or fracture healing was available.  in this work we present approximately ests from the domestic sheep. this represents an important resource to researchers interested in the sheep as a model organism and greatly expands the number of gene sequences available for ovis aries.the sequences are freely available in genenest and the public sequence databases. we have used the sequences we generated as a tool to investigate the early stages of fracture healing and have identified numerous genes that are differentially regulated in early callus tissues, many of which were not previously known to have any specific relation to fracture healing or bone development. these genes will be valuable leads for further research into the molecular mechanisms of fracture healing. 
 of the genes were differentially expressed between postfracture days and which could reflect an important role for these genes for the initiation of osteogenesis. despite these medical uses and the well known economic and cultural importance of the sheep, relatively little research has been performed into sheep genetics, and dna sequences are available for only a small number of sheep genes. we used the newly identified sequences to develop rt-pcr assays for sheep genes and measured differential expression during the course of fracture healing between days and postfracture. the sequences we have identified in this work are a valuable resource for future studies on musculoskeletal healing and regeneration using sheep and represent an important head-start for genomic sequencing projects for ovis aries, with partial or complete sequences being made available for over previously unsequenced sheep genes. all genes showed significant shifts at one or more time points. these ests were clustered with the previously available thousand sheep ests to a total of contigs with an average length of nucleotides. in this work we have sequenced over thousand expressed sequence tags from libraries developed from healing bone in a sheep model of fracture healing.
 the sheep is an important model animal for testing novel fracture treatments and other medical applications. despite these medical uses and the well known economic and cultural importance of the sheep, relatively little research has been performed into sheep genetics, and dna sequences are available for only a small number of sheep genes.  in this work we have sequenced over thousand expressed sequence tags from libraries developed from healing bone in a sheep model of fracture healing. these ests were clustered with the previously available thousand sheep ests to a total of contigs with an average length of nucleotides. we used the newly identified sequences to develop rt-pcr assays for sheep genes and measured differential expression during the course of fracture healing between days and postfracture. all genes showed significant shifts at one or more time points. of the genes were differentially expressed between postfracture days and which could reflect an important role for these genes for the initiation of osteogenesis.  the sequences we have identified in this work are a valuable resource for future studies on musculoskeletal healing and regeneration using sheep and represent an important head-start for genomic sequencing projects for ovis aries, with partial or complete sequences being made available for over previously unsequenced sheep genes. 