BACKGROUND
the development of the respiratory system represents an evolutionary hallmark that allowed vertebrates to survive on land utilizing air as a source of oxygen.
abnormal development of the respiratory system in humans is associated with multiple disorders such as tracheal/bronchial atresia, tracheo-esophageal fistula, bronchogenic cysts, pulmonary/lobar atresia, and pulmonary hypoplasia  <cit> .
in the embryonic trachea, the endoderm differentiates into a ciliated pseudo-stratified epithelium, which includes basal p63+ cells, clara cells, neuroendocrine cells, and ciliated cells.
the ventral mesenchyme gradually matures to create c-shaped cartilage rings, while the dorsal mesenchyme forms contractile cells comprising the trachealis smooth muscle, which defines the posterior boundary between the trachea and esophagus.
tracheal wall malformation can be due to congenital or acquired abnormalities.
the congenital forms are related to immature tracheal cartilage, whereas the more common acquired forms occur as a consequence of compression damage or degeneration of the tracheal cartilage, such as in vascular sling anomalies  <cit> .
tracheomalacia is a condition described as causing excessive expiratory collapse of the trachea, resulting from weakness of the supporting structures of tracheal and bronchial walls, leading to symptoms of airway obstruction  <cit> .
β-catenin, bmp <dig>  and shh are previously well-characterized regulators of cartilage formation, which may activate the sox <dig> gene in mesenchymal cells to induce their differentiation into chondrocytes.
members of the fibroblast growth factor superfamily, including fgf <dig>  fgf <dig>  and fgf <dig>  are also key regulators of cartilage formation  <cit> .
fgf <dig> regulates proliferation and spatial localization of the chondrocytes in the developing upper respiratory tract  <cit> .
tbx <dig> and tbx <dig> double knockout in lung resulted in defects of cartilage formation in mouse trachea and in altered lung branching, with tbx <dig> and tbx <dig> acting upstream of sox <dig> in tracheal cartilage development  <cit> .
here, we used an inducible tbx <dig> gene-driven system to knock out the sox <dig> gene specifically in lung mesenchyme  <cit> , in order to elucidate more fully its function within the mesenchyme during lung development.
mesenchymal sox <dig> knockout had no affect on distal lung branching, on formation of smooth muscle around the bronchi, or on the trachealis smooth muscle, whereas tracheal cartilage formation was completely inhibited.
in situ hybridization revealed that in the sox <dig> null trachea, the characteristic tbx <dig>  tbx <dig>  and fgf <dig> expression pattern was lost or severely disrupted.
mutant sox <dig> mouse tracheal epithelium was also altered: cells had a higher number of cytoplasmic vacuoles, with fewer basal cells  and more clara cells .
mutant tracheal epithelium also had fewer goblet cells compared with wild-type trachea.
surfactant proteins a and c were also downregulated.
finally, tracheal lymphatic vessels were significantly underdeveloped after the knockout of mesenchymal sox <dig>  in conclusion, mesenchymal sox9-expressing cells cover a critical developmental role in the formation of tracheal cartilage rings, and in the appropriate differentiation of lung tracheal epithelium and lymphatic system.
RESULTS
sox <dig> expression during lung development
we used western blot to analyze the expression of sox <dig> in mouse lung during development.
sox <dig> protein expression level is high in embryonic lungs but drastically decreases in mouse lung after birth.
adult mouse lung does not express sox <dig> protein at all .
at embryonic day  <dig> , sox <dig> is expressed by distal lung epithelial cells and by a population of mesenchymal cells surrounding the trachea, bronchi, and bronchioles.
at e <dig> , mesenchymal cells expressing sox <dig> cells are condensed at the area of the future cartilage rings, while epithelial cells expressing sox <dig> are localized in the distal lung epithelium .
to specifically knock out sox <dig> in mesenchymal cells, we used a tbx4-rtta/tet-on-cre system, in which rtta gene expression is regulated by the mesenchymal specific enhancer of the tbx <dig> gene  <cit> .
tbx <dig> is expressed by tracheal mesenchyme and most of the lung distal mesenchyme .
sox <dig> is efficiently deleted in the tbx4-rtta/tet-on-cre/sox9fl/fl at e <dig>  and e <dig>  stage .
mesenchymal sox <dig> knockout mice showed an absence of tracheal cartilage rings
perl reported that epithelial sox <dig> deletion did not affect lung development  <cit> .
however, more recently, chang showed that sox <dig> deletion in lung epithelium resulted in a smaller lung, with fewer and dilated airway branches  <cit> .
it is possible that the use of different mouse genetic backgrounds is the cause of these contrasting results.
the role of mesenchymal sox <dig> expression is unknown.
in our study, transgenic mice lacking mesenchymal sox <dig> expression were born at the expected mendelian frequency and appeared normal at birth, but rapidly became cyanotic and showed marked signs of respiratory obstruction including gasping and retractions ; moreover, not a single transgenic mouse lived more than a few hours after birth .
despite the lethal respiratory obstruction phenotype, no difference in weight at birth was observed .
at both e <dig>  and e <dig> , embryonic lung branching did not appear to be affected by lack of mesenchymal sox <dig> expression .
however, transgenic embryos were missing cartilage rings around the trachea and bronchi.
.
transverse sections of transgenic embryo e <dig>  tracheas showed collapsed airway lumen, and a shape and appearance similar to esophagus; however, the esophagus was still present.
in 8% of the embryos, rudimentary cartilage spots developed on the ventral side of the trachea .
these spots were still expressing sox <dig> protein, indicating the existence of another unique population of sox9+ cells that either did not continue to express the tbx <dig> gene or did not express the transgene, or expressed the transgene, but did not recombine .
lung branching was not affected by lack of mesenchyme sox <dig> expression , and differentiation of distal lung epithelium and mesenchyme appeared to be normal in the mutants .
the tracheal phenotype depends on the timing of doxycycline exposure
the observed phenotype  was obtained by knocking out the sox <dig> gene in the mesenchyme of the lung, starting from day e <dig>  to the day of collection of the embryos .
we then exposed pregnant time-mated females to different durations of doxycycline induction to determine the relation between phenotype and doxycycline induction .
shorter exposure to doxycycline resulted in a milder phenotype , as embryos exposed to doxycycline for shorter time periods developed some of the more distal cartilage rings.
if doxycycline was removed from the food at e <dig> , the two most proximal cartilage rings were fully formed .
sox <dig> knockout in e <dig>  embryos did not induce any abnormalities in the cartilage development, as tbx <dig> and sox <dig> protein are not co-expressed in the tracheal mesenchyme at that stage .
expression pattern of tbx <dig>  tbx <dig>  and fgf <dig> is altered in sox <dig> mutant trachea
we next determined whether the expression pattern of genes known to be involved in tracheal cartilage ring formation was altered in the mutant trachea.
tbx <dig> and tbx <dig> are expressed between the tracheal cartilage rings in normal mouse trachea  <cit> .
in situ hybridization on e <dig>  embryonic lung tissue revealed that tbx <dig> and tbx <dig> expression patterning was lost in the mutant trachea .
tbx <dig> and tbx <dig> were expressed uniformly by all the mesenchyme lining the trachea , instead of there being an alternating striped pattern as observed in the control trachea .
using real-time reverse transcription quantitative pcr  on rna extracted from e <dig>  tracheas, we observed increased expression of tbx <dig> and tbx <dig> in the mutant trachea versus the control trachea .
fgf <dig> is another known key regulator of tracheal cartilage development.
the fgf <dig> expression pattern in the e <dig>  stage trachea normally delimits the primitive cartilage rings; however, this specific expression pattern was lost after sox <dig> knockout, and fgf <dig> was expressed by all the tracheal mesenchyme .
we observed a trend towards decreased fgf <dig> mrna expression in the mutant trachea but it was not statistically significant.
trachealis contractile muscle pattern formation and bronchial smooth muscle cells are not affected by sox <dig> knockout
the tbx <dig>  tbx <dig>  and fgf <dig> expression patterns were lost in sox <dig> mutants.
this observation led us to investigate whether the sox <dig> gene is also necessary for patterning the sm-22-α + cells, which are located at the junction between the trachea and esophagus , and form the trachealis muscle.
because sox9-expressing cells are located at the ventral region of the trachea, sm-22-α and sox <dig> expression seems to be mutually exclusive, suggesting that expression of sox <dig> is limiting the expression of sm-22-α to the dorsal area of the trachea, and vice versa.
however, we found that sox <dig> expression is not involved in patterning the expression of sm-22-α-expressing cells in the trachea .
in fact, the contractile lunar-shaped ring of sm-22-α + cells was still present and localized in the dorsal area of the mutant trachea.
the absolute area covered by sm22-α + cells did not change significantly in the mutant trachea versus the wild-type trachea .
we also analyzed whether the sox <dig> gene was involved in the proliferation of bronchial smooth muscle cells; however, we did not observe any obvious change in smooth muscle cells lining the bronchi of mutant mouse lung .
tracheal epithelium is altered after sox <dig> knockout
mice lacking sox <dig> in the lung mesenchyme died at birth, due to an inability to breathe because of the collapsed trachea.
no change in smooth muscle cell numbers or patterning was observed.
however, the epithelium lining the trachea was abnormal; epithelial cells in the mutant trachea had larger and more numerous vacuoles .
electron microscopy revealed that some of those cells looked like clara cells .
real-time rt-qpcr was used to quantify change of expression of lung-relevant genes in mutant mouse trachea.
expression of the surfactant protein genes sftp-a and sftp-c mrna was reduced in the sox9Δ/Δ mice, and cc <dig> mrna was slightly overexpressed in the mutant trachea , indicating altered differentiation of tracheal epithelium.
the number of ciliated cells did not change after mesenchymal deletion of the sox <dig> gene .
however, at e <dig> , the number of tracheal basal cells  decreased  and clara cells   increased in the mutant trachea versus the control trachea.
e <dig>  sox9fl/fl and sox9∆/∆ tracheas showed a similar number of p63+ cells .
alcian blue staining of transverse sections of trachea revealed decreased mucin production by the mutant tracheal epithelium .
agr <dig> protein, a marker of lung goblet cells, was downregulated in the tracheal epithelium of the sox9Δ/Δ embryos compared with the sox9fl/fl embryos .
however, expression of sox <dig> and foxp <dig>  key regulators of lung epithelium differentiation, was unaltered after sox <dig> knock out .
despite the altered differentiation, proliferation of tracheal epithelium was not affected by lack of mesenchymal sox <dig> expression, no change in phospho-histone- <dig>  and total cell number per tracheal transversal section was found .
tracheal lymphatic system was altered in sox9Δ/Δ trachea
we quantified the area covered by lung mesenchyme  and the mesenchyme cell number on transverse sections of e <dig>  wild-type and mutant tracheas.
despite the somewhat misleading visual appearance, both the mesenchyme cell numbers and the surface covered by them were the same in the sox9fl/fl and sox9Δ/Δ trachea .
prox <dig> and lyve <dig> are key markers of lymphatic vessel development  <cit> .
prox <dig> also controls neuronal progenitor cell differentiation to maintain the required balance between different cell types  <cit> .
we found that the tracheal lymphatic system was significantly underdeveloped in the mutant sox <dig> embryos, as shown by reduced immunostaining of prox <dig>  lyve <dig>  and pecam .
in addition, the, mrna level of prox <dig>  lyve <dig>  and vegfr <dig> was reduced in the sox9Δ/Δ tracheas .
discussion
normal lung branching is achieved by tightly regulated crosstalk between mesenchymal and epithelial compartments.
specific growth factors are released from one compartment to activate expression of genes in the other compartment, and vice versa <cit> .
sox <dig> is a multifunctional transcriptional factor expressed in both embryonic lung mesenchyme and epithelium.
perl et al.
reported that deletion of sox <dig> gene in embryonic lung epithelium did not affect lung development; mice lacking sox <dig> in the lung epithelium were viable and fertile, and did not show any differences from their control littermate, even when exposed to hyperoxia-induced injury  <cit> .
however, more recently, chang et al.
showed that sox <dig> is necessary for proper lung branching  <cit> .
sox <dig> is also a key regulator of embryonic kidney epithelial branching  <cit> .
these contrasting reports on sox <dig> regulation of lung epithelial lung branching may be due to the use of different mouse genetic backgrounds by perl and chang.
here, we focused on the role of the mesenchymal sox9-expressing cells, which are localized around the trachea and main bronchi.
by using an inducible doxycycline-regulated system, driven by part of the tbx <dig> gene promoter  <cit> , we were able to delete sox <dig> expression specifically in the mesenchymal compartment of the murine lung.
our collaborator shi  <cit>  developed the tbx4-rtta transgenic mouse line and in the present study, we showed that this mouse line specifically deleted sox <dig> in lung mesenchyme  and likewise activated a green fluorescent protein  reporter gene in lung mesenchyme .
mice lacking sox <dig> did not develop tracheal cartilage rings, were unable to breathe because of tracheal collapse, and died shortly after birth.
transgenic mice appeared normal at birth, but then rapidly showed clear evidence of cyanosis , and not a single transgenic pup lived more than a few hours after birth , despite the birth weights of the mutants being similar to those of wild-type mice .
these data suggest that lack of mesenchymal sox <dig> in embryonic lung is not compatible with life.
arora et al.
obtained a similar but milder tracheal cartilage phenotype in transgenic mice missing both the tbx <dig> and tbx <dig> genes; some cartilage trachea rings still developed, but they were smaller and had an abnormal shape  <cit> .
a tbx <dig> and tbx <dig> combined deletion also resulted in altered lung branching.
data from the same laboratory also suggested that tbx <dig> and tbx <dig> are upstream regulatory genes of sox <dig> in the embryonic lung mesenchyme  <cit> .
lung mesenchyme sox <dig> knockout mice did not show an obvious phenotype on lung branching; the number of terminal branches of the left lobe of e <dig>  lungs was not different between the wild-type and mutant embryos .
moreover, differentiation of distal lung epithelium and mesenchyme was not affected by lack of sox <dig> mesenchymal expression at e <dig>  .
thus, sox <dig> deletion appears to affect only tracheal development.
in our mesenchyme-specific sox <dig> knockout model, tbx <dig> and tbx <dig> expression was enhanced in the trachea.
this indicates the existence of a complex feedback from tbx4/ <dig> to sox <dig> and vice versa.
thus, tbx4/ <dig> gene expression may have been upregulated, and may thus have partially compensated for the lost sox <dig> expression in our mutant.
moreover, sox <dig> knockout mice also had an altered tbx4/ <dig> pattern of expression.
in normal lung trachea, the tbx <dig> and tbx <dig> genes are expressed by the mesenchyme in the intervals between the developing cartilage rings.
thus, the tbx4/ <dig> genes and the sox <dig> gene are normally mutually exclusive in the tracheal mesenchyme at e <dig> .
when the sox <dig> gene was knocked out, the tbx4/ <dig> genes were uniformly expressed by all the mesenchyme surrounding the trachea.
a similar result was obtained with fgf <dig> pattern expression.
at e <dig> , fgf <dig> is normally expressed by the developing cartilage rings in the trachea.
inactivation of sox <dig> resulted in the disruption of the normal fgf <dig> spatial expression pattern; fgf <dig> was now expressed by all the mesenchyme surrounding the trachea.
quantification of the mrna of fgf <dig> did show a small trend towards a decrease in mrna level, but this did not reach statistical significance.
taken together, these data clearly indicate that the sox <dig> gene has a key role in patterning the expression of tbx4/ <dig> and fgf <dig> in the embryonic trachea.
genes linking sox <dig> expression to tbx4/ <dig> and fgf <dig> gene expression pattern will be the subject of future studies.
the tbx4-rtta inducible system allowed us to closely regulate the timing of sox <dig> deletion.
we took advantage of this to investigate how the length of time of doxycycline induction relates to the severity of the phenotype.
we found that shorter induction resulted in a milder phenotype.
if the doxycycline was removed from the food at e <dig> , the two most distal cartilage rings formed fully , but in these mice, the tbx <dig> gene was expressed by all the lung mesenchyme  <cit> , which implies that all lung mesenchymal cells in these mice have a non-functional sox <dig> gene at that stage.
there are several possible explanations for these results.
it could be that mesenchymal sox9-positive cells are continuously migrating from the laryngeal region into the tracheal mesenchyme to contribute to cartilage ring formation, or that the shorter doxycycline exposure results in incomplete sox <dig> deletion.
another possible explanation is that tbx4-rtta is expressed in distal mesenchyme and then proceeds towards the larynx over time.
future studies involving the use of a sox9-er-cre mouse line in combination with our tbx4-rtta model should be able to determine which explanation is likely to be the correct one.
however, considering that sox <dig> is also expressed by neural crest cells  <cit> , which are ‘migratory’ cells, the hypothesis of sox <dig> continuously migrating into the lung mesenchyme seems to be the most plausible, and it will be the first hypothesis to be tested in our future studies.
by e <dig>  to e <dig> , tracheal tbx <dig> and sox <dig> are mutually exclusively expressed, thus as predicted, knockout of the sox <dig> gene atat this later stage  did not affect tracheal cartilage development .
the ability to regulate the severity of the tracheal phenotype will allow us to tailor timed knockouts to model specific tracheal and lung disease models for future studies.
as pointed out above, mesenchymal-epithelial crosstalk is required for correct development of the embryonic lung.
therefore, anatomical or molecular alterations of one embryonic compartment such as mesenchyme may affect the correct development of the epithelia, and vice versa.
we noticed that the tracheal epithelium was altered after mesenchymal sox <dig> knockout.
cell count analysis revealed that mutant trachea had a lower number of basal p63+ cells, and a higher number of clara  cells.
the number of ciliated cells did not change.
an immediate conclusion would be that the absence of cartilage rings around the embryonic trachea caused the tracheal epithelium to behave more similarly to the more distal intralobar bronchiolar epithelium.
as the number of basal cells was not altered in the e <dig>  sox9Δ/Δ trachea, we suggest that basal cell differentiation is not directly dependent on sox <dig> expression but is more likely to depend on cartilage condensation, which normally starts to occur at e <dig> .
thus, we speculate that a specific growth factor signal  may be released by the cartilage rings to promote correct tracheal epithelium differentiation.
sox9Δ/Δ trachea had reduced production of agr <dig>  a protein that identifies goblet cells in the lung.
moreover, transcripts of both surfactant proteins a and c were reduced in the mutant trachea, further supporting the role of sox <dig> expressing cells in the differentiation of tracheal epithelium.
despite the altered differentiation, proliferation of tracheal epithelium was not affected by lack of mesenchymal sox <dig> expression.
no change in ph <dig> or epithelial cell number per transversal section was found .
thus, lack of tracheal rings altered proper differentiation of tracheal epithelium but not its proliferation.
the expression pattern of fgf <dig> in sox9Δ/Δ embryonic trachea was markedly altered.
even though it was not statistically significant, fgf <dig> mrna level was slightly decreased in the mutant trachea.
volckaert et al.
<cit>  showed that fgf <dig> overexpression resulted in an increased number of basal cells and decreased number of clara cells  <cit> .
because of the concordance of these and our results, we hypothesize that incorrect expression of fgf <dig> may be mostly responsible for the altered differentiation of tracheal epithelium in the sox9Δ/Δ trachea.
we cannot conclude without any doubt that the epithelial phenotype is directly related to the absence of signals originating from the cartilage rings.
an alternative plausible model is that the lack of mechanical support  of the trachea instructs the epithelium to differentiate onto a different path.
finally, the lack of proper vasculature development  in the mutant trachea may also contribute to the altered differentiation of the tracheal epithelium.
gli2–/–,gli3+/– mutants displayed esophageal atresia with tracheo-esophageal fistula and a severe lung phenotype  <cit> .
gli <dig> and gli <dig> gene expression in the sox9Δ/Δ trachea did not change in our sox <dig> knockout model .
shh and gli <dig> are also key regulators of cartilage development  <cit> , but their expression was not altered in our sox9Δ/Δ embryonic trachea .
expression of collagen <dig>  a key regulator of cartilage differentiation, was reduced after sox <dig> deletion, supporting the model in which sox <dig> acts upstream of collagen <dig> to control tracheal cartilage development.
despite the somewhat misleading visual appearance, the numbers of mesenchymal cells and the area covered by them were not different between the sox9fl/fl and sox9Δ/Δ trachea .
thus, the apparent difference in size observed between the wild-type and mutant tracheas is caused by the lack of the cartilage rings in the sox9Δ/Δ trachea.
pecam is broadly expressed by vascular structures  <cit> .
and its expression in the distal lung mesenchyme was assayed to investigate any alterations of distal mesenchyme lung differentiation.
as we observed for the distal epithelium, the distal lung mesenchyme appeared normal in the mutant trachea.
we concluded that lack of sox <dig> in the lung mesenchyme did not affect the differentiation or development of the vasculature in the distal lung mesenchyme.
the lymphatic vascular system serves to transport tissue fluid, extravasated plasma proteins, and cells back to the circulation  <cit> .
prox <dig> and lyve <dig> are key regulators and markers of lymphatic vessel development.
the lymphatic system was significantly underdeveloped in the mutant sox <dig> tracheas; immunostaining of prox <dig>  lyve <dig>  and pecam, and mrna level of prox <dig>  lyve <dig>  and vegfr <dig> were reduced in the sox9Δ/Δ tracheas.
thus, lack of tracheal cartilage affects appropriate tracheal lymphatic vessel development.
it is also possible that an early sox9-expressing mesenchymal cell population exists, and that this differentiates into lymphatic vessel cells.
as pecam is expressed by both blood and lymphatic vascular vessels, we speculate that mesenchymal sox <dig> expression has a broad role in the generalized vascularization of the trachea.
the mechanism linking the lack of cartilage rings to the altered tracheal vascularization is under further investigation.
we focused on epidermal growth factor, as it is known to be involved in lymphatic vessel modeling in the skin  <cit> , and found it to be downregulated in the sox9Δ/Δ trachea .
CONCLUSIONS
in conclusion, we have uncovered a key fundamental role of the mesenchymal sox9-expressing cells in the development of the tracheal cartilage rings, which in turn induce proper tracheal epithelium differentiation and lymphatic system development .
thus, besides having a mechanical function, tracheal cartilage also appears to be a local signaling structure in the embryonic lung.
some pieces of this puzzle are still missing, and are currently under investigation.
nevertheless, we have identified an important mechanism by which mesenchymal sox9-expressing cells instruct tracheal cartilage, epithelial, and lymphatic differentiation in a phenotype that already appears to model and may perhaps explain mechanistically some extreme cases of primary tracheomalacia in human new born infants.
the appropriate temporospatial expression of sox <dig> in lung mesenchyme is necessary for correct tracheal cartilage formation, lymphatic vasculature system development, and epithelial differentiation.
our study showed that a lack of tracheal cartilage rings resulted in increase in clara cells and decrease in basal and goblet cells in the tracheal epithelium.
we also uncovered a novel mechanism of lung epithelium differentiation; tracheal cartilage rings instruct the tracheal epithelium to differentiate properly during embryonic development.
thus, besides having a mechanical function, tracheal cartilage also appears to be a local signaling structure in the embryonic lung.

 the development of the respiratory system represents an evolutionary hallmark that allowed vertebrates to survive on land utilizing air as a source of oxygen. abnormal development of the respiratory system in humans is associated with multiple disorders such as tracheal/bronchial atresia, tracheo-esophageal fistula, bronchogenic cysts, pulmonary/lobar atresia, and pulmonary hypoplasia. in the embryonic trachea, the endoderm differentiates into a ciliated pseudo-stratified epithelium, which includes basal p63+ cells, clara cells, neuroendocrine cells, and ciliated cells. the ventral mesenchyme gradually matures to create c-shaped cartilage rings, while the dorsal mesenchyme forms contractile cells comprising the trachealis smooth muscle, which defines the posterior boundary between the trachea and esophagus. tracheal wall malformation can be due to congenital or acquired abnormalities. the congenital forms are related to immature tracheal cartilage, whereas the more common acquired forms occur as a consequence of compression damage or degeneration of the tracheal cartilage, such as in vascular sling anomalies. tracheomalacia is a condition described as causing excessive expiratory collapse of the trachea, resulting from weakness of the supporting structures of tracheal and bronchial walls, leading to symptoms of airway obstruction. β-catenin, bmp and shh are previously well-characterized regulators of cartilage formation, which may activate the sox gene in mesenchymal cells to induce their differentiation into chondrocytes. members of the fibroblast growth factor superfamily, including fgf fgf and fgf are also key regulators of cartilage formation. fgf regulates proliferation and spatial localization of the chondrocytes in the developing upper respiratory tract. tbx and tbx double knockout in lung resulted in defects of cartilage formation in mouse trachea and in altered lung branching, with tbx and tbx acting upstream of sox in tracheal cartilage development. here, we used an inducible tbx gene-driven system to knock out the sox gene specifically in lung mesenchyme, in order to elucidate more fully its function within the mesenchyme during lung development. mesenchymal sox knockout had no affect on distal lung branching, on formation of smooth muscle around the bronchi, or on the trachealis smooth muscle, whereas tracheal cartilage formation was completely inhibited. in situ hybridization revealed that in the sox null trachea, the characteristic tbx tbx and fgf expression pattern was lost or severely disrupted. mutant sox mouse tracheal epithelium was also altered: cells had a higher number of cytoplasmic vacuoles, with fewer basal cells and more clara cells. mutant tracheal epithelium also had fewer goblet cells compared with wild-type trachea. surfactant proteins a and c were also downregulated. finally, tracheal lymphatic vessels were significantly underdeveloped after the knockout of mesenchymal sox in conclusion, mesenchymal sox9-expressing cells cover a critical developmental role in the formation of tracheal cartilage rings, and in the appropriate differentiation of lung tracheal epithelium and lymphatic system.  sox expression during lung development we used western blot to analyze the expression of sox in mouse lung during development. sox protein expression level is high in embryonic lungs but drastically decreases in mouse lung after birth. adult mouse lung does not express sox protein at all. at embryonic day, sox is expressed by distal lung epithelial cells and by a population of mesenchymal cells surrounding the trachea, bronchi, and bronchioles. at e, mesenchymal cells expressing sox cells are condensed at the area of the future cartilage rings, while epithelial cells expressing sox are localized in the distal lung epithelium. to specifically knock out sox in mesenchymal cells, we used a tbx4-rtta/tet-on-cre system, in which rtta gene expression is regulated by the mesenchymal specific enhancer of the tbx gene. tbx is expressed by tracheal mesenchyme and most of the lung distal mesenchyme. sox is efficiently deleted in the tbx4-rtta/tet-on-cre/sox9fl/fl at e and e stage. mesenchymal sox knockout mice showed an absence of tracheal cartilage rings perl reported that epithelial sox deletion did not affect lung development. however, more recently, chang showed that sox deletion in lung epithelium resulted in a smaller lung, with fewer and dilated airway branches. it is possible that the use of different mouse genetic backgrounds is the cause of these contrasting results. the role of mesenchymal sox expression is unknown. in our study, transgenic mice lacking mesenchymal sox expression were born at the expected mendelian frequency and appeared normal at birth, but rapidly became cyanotic and showed marked signs of respiratory obstruction including gasping and retractions; moreover, not a single transgenic mouse lived more than a few hours after birth. despite the lethal respiratory obstruction phenotype, no difference in weight at birth was observed. at both e and e, embryonic lung branching did not appear to be affected by lack of mesenchymal sox expression. however, transgenic embryos were missing cartilage rings around the trachea and bronchi.. transverse sections of transgenic embryo e tracheas showed collapsed airway lumen, and a shape and appearance similar to esophagus; however, the esophagus was still present. in 8% of the embryos, rudimentary cartilage spots developed on the ventral side of the trachea. these spots were still expressing sox protein, indicating the existence of another unique population of sox9+ cells that either did not continue to express the tbx gene or did not express the transgene, or expressed the transgene, but did not recombine. lung branching was not affected by lack of mesenchyme sox expression, and differentiation of distal lung epithelium and mesenchyme appeared to be normal in the mutants. the tracheal phenotype depends on the timing of doxycycline exposure the observed phenotype was obtained by knocking out the sox gene in the mesenchyme of the lung, starting from day e to the day of collection of the embryos. we then exposed pregnant time-mated females to different durations of doxycycline induction to determine the relation between phenotype and doxycycline induction. shorter exposure to doxycycline resulted in a milder phenotype, as embryos exposed to doxycycline for shorter time periods developed some of the more distal cartilage rings. if doxycycline was removed from the food at e, the two most proximal cartilage rings were fully formed. sox knockout in e embryos did not induce any abnormalities in the cartilage development, as tbx and sox protein are not co-expressed in the tracheal mesenchyme at that stage. expression pattern of tbx tbx and fgf is altered in sox mutant trachea we next determined whether the expression pattern of genes known to be involved in tracheal cartilage ring formation was altered in the mutant trachea. tbx and tbx are expressed between the tracheal cartilage rings in normal mouse trachea. in situ hybridization on e embryonic lung tissue revealed that tbx and tbx expression patterning was lost in the mutant trachea. tbx and tbx were expressed uniformly by all the mesenchyme lining the trachea, instead of there being an alternating striped pattern as observed in the control trachea. using real-time reverse transcription quantitative pcr on rna extracted from e tracheas, we observed increased expression of tbx and tbx in the mutant trachea versus the control trachea. fgf is another known key regulator of tracheal cartilage development. the fgf expression pattern in the e stage trachea normally delimits the primitive cartilage rings; however, this specific expression pattern was lost after sox knockout, and fgf was expressed by all the tracheal mesenchyme. we observed a trend towards decreased fgf mrna expression in the mutant trachea but it was not statistically significant. trachealis contractile muscle pattern formation and bronchial smooth muscle cells are not affected by sox knockout the tbx tbx and fgf expression patterns were lost in sox mutants. this observation led us to investigate whether the sox gene is also necessary for patterning the sm-22-α + cells, which are located at the junction between the trachea and esophagus, and form the trachealis muscle. because sox9-expressing cells are located at the ventral region of the trachea, sm-22-α and sox expression seems to be mutually exclusive, suggesting that expression of sox is limiting the expression of sm-22-α to the dorsal area of the trachea, and vice versa. however, we found that sox expression is not involved in patterning the expression of sm-22-α-expressing cells in the trachea. in fact, the contractile lunar-shaped ring of sm-22-α + cells was still present and localized in the dorsal area of the mutant trachea. the absolute area covered by sm22-α + cells did not change significantly in the mutant trachea versus the wild-type trachea. we also analyzed whether the sox gene was involved in the proliferation of bronchial smooth muscle cells; however, we did not observe any obvious change in smooth muscle cells lining the bronchi of mutant mouse lung. tracheal epithelium is altered after sox knockout mice lacking sox in the lung mesenchyme died at birth, due to an inability to breathe because of the collapsed trachea. no change in smooth muscle cell numbers or patterning was observed. however, the epithelium lining the trachea was abnormal; epithelial cells in the mutant trachea had larger and more numerous vacuoles. electron microscopy revealed that some of those cells looked like clara cells. real-time rt-qpcr was used to quantify change of expression of lung-relevant genes in mutant mouse trachea. expression of the surfactant protein genes sftp-a and sftp-c mrna was reduced in the sox9Δ/Δ mice, and cc mrna was slightly overexpressed in the mutant trachea, indicating altered differentiation of tracheal epithelium. the number of ciliated cells did not change after mesenchymal deletion of the sox gene. however, at e, the number of tracheal basal cells decreased and clara cells increased in the mutant trachea versus the control trachea. e sox9fl/fl and sox9∆/∆ tracheas showed a similar number of p63+ cells. alcian blue staining of transverse sections of trachea revealed decreased mucin production by the mutant tracheal epithelium. agr protein, a marker of lung goblet cells, was downregulated in the tracheal epithelium of the sox9Δ/Δ embryos compared with the sox9fl/fl embryos. however, expression of sox and foxp key regulators of lung epithelium differentiation, was unaltered after sox knock out. despite the altered differentiation, proliferation of tracheal epithelium was not affected by lack of mesenchymal sox expression, no change in phospho-histone- and total cell number per tracheal transversal section was found. tracheal lymphatic system was altered in sox9Δ/Δ trachea we quantified the area covered by lung mesenchyme and the mesenchyme cell number on transverse sections of e wild-type and mutant tracheas. despite the somewhat misleading visual appearance, both the mesenchyme cell numbers and the surface covered by them were the same in the sox9fl/fl and sox9Δ/Δ trachea. prox and lyve are key markers of lymphatic vessel development. prox also controls neuronal progenitor cell differentiation to maintain the required balance between different cell types. we found that the tracheal lymphatic system was significantly underdeveloped in the mutant sox embryos, as shown by reduced immunostaining of prox lyve and pecam. in addition, the, mrna level of prox lyve and vegfr was reduced in the sox9Δ/Δ tracheas. discussion normal lung branching is achieved by tightly regulated crosstalk between mesenchymal and epithelial compartments. specific growth factors are released from one compartment to activate expression of genes in the other compartment, and vice versa. sox is a multifunctional transcriptional factor expressed in both embryonic lung mesenchyme and epithelium. perl et al. reported that deletion of sox gene in embryonic lung epithelium did not affect lung development; mice lacking sox in the lung epithelium were viable and fertile, and did not show any differences from their control littermate, even when exposed to hyperoxia-induced injury. however, more recently, chang et al. showed that sox is necessary for proper lung branching. sox is also a key regulator of embryonic kidney epithelial branching. these contrasting reports on sox regulation of lung epithelial lung branching may be due to the use of different mouse genetic backgrounds by perl and chang. here, we focused on the role of the mesenchymal sox9-expressing cells, which are localized around the trachea and main bronchi. by using an inducible doxycycline-regulated system, driven by part of the tbx gene promoter, we were able to delete sox expression specifically in the mesenchymal compartment of the murine lung. our collaborator shi developed the tbx4-rtta transgenic mouse line and in the present study, we showed that this mouse line specifically deleted sox in lung mesenchyme and likewise activated a green fluorescent protein reporter gene in lung mesenchyme. mice lacking sox did not develop tracheal cartilage rings, were unable to breathe because of tracheal collapse, and died shortly after birth. transgenic mice appeared normal at birth, but then rapidly showed clear evidence of cyanosis, and not a single transgenic pup lived more than a few hours after birth, despite the birth weights of the mutants being similar to those of wild-type mice. these data suggest that lack of mesenchymal sox in embryonic lung is not compatible with life. arora et al. obtained a similar but milder tracheal cartilage phenotype in transgenic mice missing both the tbx and tbx genes; some cartilage trachea rings still developed, but they were smaller and had an abnormal shape. a tbx and tbx combined deletion also resulted in altered lung branching. data from the same laboratory also suggested that tbx and tbx are upstream regulatory genes of sox in the embryonic lung mesenchyme. lung mesenchyme sox knockout mice did not show an obvious phenotype on lung branching; the number of terminal branches of the left lobe of e lungs was not different between the wild-type and mutant embryos. moreover, differentiation of distal lung epithelium and mesenchyme was not affected by lack of sox mesenchymal expression at e. thus, sox deletion appears to affect only tracheal development. in our mesenchyme-specific sox knockout model, tbx and tbx expression was enhanced in the trachea. this indicates the existence of a complex feedback from tbx4/ to sox and vice versa. thus, tbx4/ gene expression may have been upregulated, and may thus have partially compensated for the lost sox expression in our mutant. moreover, sox knockout mice also had an altered tbx4/ pattern of expression. in normal lung trachea, the tbx and tbx genes are expressed by the mesenchyme in the intervals between the developing cartilage rings. thus, the tbx4/ genes and the sox gene are normally mutually exclusive in the tracheal mesenchyme at e. when the sox gene was knocked out, the tbx4/ genes were uniformly expressed by all the mesenchyme surrounding the trachea. a similar result was obtained with fgf pattern expression. at e, fgf is normally expressed by the developing cartilage rings in the trachea. inactivation of sox resulted in the disruption of the normal fgf spatial expression pattern; fgf was now expressed by all the mesenchyme surrounding the trachea. quantification of the mrna of fgf did show a small trend towards a decrease in mrna level, but this did not reach statistical significance. taken together, these data clearly indicate that the sox gene has a key role in patterning the expression of tbx4/ and fgf in the embryonic trachea. genes linking sox expression to tbx4/ and fgf gene expression pattern will be the subject of future studies. the tbx4-rtta inducible system allowed us to closely regulate the timing of sox deletion. we took advantage of this to investigate how the length of time of doxycycline induction relates to the severity of the phenotype. we found that shorter induction resulted in a milder phenotype. if the doxycycline was removed from the food at e, the two most distal cartilage rings formed fully, but in these mice, the tbx gene was expressed by all the lung mesenchyme, which implies that all lung mesenchymal cells in these mice have a non-functional sox gene at that stage. there are several possible explanations for these results. it could be that mesenchymal sox9-positive cells are continuously migrating from the laryngeal region into the tracheal mesenchyme to contribute to cartilage ring formation, or that the shorter doxycycline exposure results in incomplete sox deletion. another possible explanation is that tbx4-rtta is expressed in distal mesenchyme and then proceeds towards the larynx over time. future studies involving the use of a sox9-er-cre mouse line in combination with our tbx4-rtta model should be able to determine which explanation is likely to be the correct one. however, considering that sox is also expressed by neural crest cells, which are ‘migratory’ cells, the hypothesis of sox continuously migrating into the lung mesenchyme seems to be the most plausible, and it will be the first hypothesis to be tested in our future studies. by e to e, tracheal tbx and sox are mutually exclusively expressed, thus as predicted, knockout of the sox gene atat this later stage did not affect tracheal cartilage development. the ability to regulate the severity of the tracheal phenotype will allow us to tailor timed knockouts to model specific tracheal and lung disease models for future studies. as pointed out above, mesenchymal-epithelial crosstalk is required for correct development of the embryonic lung. therefore, anatomical or molecular alterations of one embryonic compartment such as mesenchyme may affect the correct development of the epithelia, and vice versa. we noticed that the tracheal epithelium was altered after mesenchymal sox knockout. cell count analysis revealed that mutant trachea had a lower number of basal p63+ cells, and a higher number of clara cells. the number of ciliated cells did not change. an immediate conclusion would be that the absence of cartilage rings around the embryonic trachea caused the tracheal epithelium to behave more similarly to the more distal intralobar bronchiolar epithelium. as the number of basal cells was not altered in the e sox9Δ/Δ trachea, we suggest that basal cell differentiation is not directly dependent on sox expression but is more likely to depend on cartilage condensation, which normally starts to occur at e. thus, we speculate that a specific growth factor signal may be released by the cartilage rings to promote correct tracheal epithelium differentiation. sox9Δ/Δ trachea had reduced production of agr a protein that identifies goblet cells in the lung. moreover, transcripts of both surfactant proteins a and c were reduced in the mutant trachea, further supporting the role of sox expressing cells in the differentiation of tracheal epithelium. despite the altered differentiation, proliferation of tracheal epithelium was not affected by lack of mesenchymal sox expression. no change in ph or epithelial cell number per transversal section was found. thus, lack of tracheal rings altered proper differentiation of tracheal epithelium but not its proliferation. the expression pattern of fgf in sox9Δ/Δ embryonic trachea was markedly altered. even though it was not statistically significant, fgf mrna level was slightly decreased in the mutant trachea. volckaert et al. showed that fgf overexpression resulted in an increased number of basal cells and decreased number of clara cells. because of the concordance of these and our results, we hypothesize that incorrect expression of fgf may be mostly responsible for the altered differentiation of tracheal epithelium in the sox9Δ/Δ trachea. we cannot conclude without any doubt that the epithelial phenotype is directly related to the absence of signals originating from the cartilage rings. an alternative plausible model is that the lack of mechanical support of the trachea instructs the epithelium to differentiate onto a different path. finally, the lack of proper vasculature development in the mutant trachea may also contribute to the altered differentiation of the tracheal epithelium. gli2–/–,gli3+/– mutants displayed esophageal atresia with tracheo-esophageal fistula and a severe lung phenotype. gli and gli gene expression in the sox9Δ/Δ trachea did not change in our sox knockout model. shh and gli are also key regulators of cartilage development, but their expression was not altered in our sox9Δ/Δ embryonic trachea. expression of collagen a key regulator of cartilage differentiation, was reduced after sox deletion, supporting the model in which sox acts upstream of collagen to control tracheal cartilage development. despite the somewhat misleading visual appearance, the numbers of mesenchymal cells and the area covered by them were not different between the sox9fl/fl and sox9Δ/Δ trachea. thus, the apparent difference in size observed between the wild-type and mutant tracheas is caused by the lack of the cartilage rings in the sox9Δ/Δ trachea. pecam is broadly expressed by vascular structures. and its expression in the distal lung mesenchyme was assayed to investigate any alterations of distal mesenchyme lung differentiation. as we observed for the distal epithelium, the distal lung mesenchyme appeared normal in the mutant trachea. we concluded that lack of sox in the lung mesenchyme did not affect the differentiation or development of the vasculature in the distal lung mesenchyme. the lymphatic vascular system serves to transport tissue fluid, extravasated plasma proteins, and cells back to the circulation. prox and lyve are key regulators and markers of lymphatic vessel development. the lymphatic system was significantly underdeveloped in the mutant sox tracheas; immunostaining of prox lyve and pecam, and mrna level of prox lyve and vegfr were reduced in the sox9Δ/Δ tracheas. thus, lack of tracheal cartilage affects appropriate tracheal lymphatic vessel development. it is also possible that an early sox9-expressing mesenchymal cell population exists, and that this differentiates into lymphatic vessel cells. as pecam is expressed by both blood and lymphatic vascular vessels, we speculate that mesenchymal sox expression has a broad role in the generalized vascularization of the trachea. the mechanism linking the lack of cartilage rings to the altered tracheal vascularization is under further investigation. we focused on epidermal growth factor, as it is known to be involved in lymphatic vessel modeling in the skin, and found it to be downregulated in the sox9Δ/Δ trachea.  in conclusion, we have uncovered a key fundamental role of the mesenchymal sox9-expressing cells in the development of the tracheal cartilage rings, which in turn induce proper tracheal epithelium differentiation and lymphatic system development. thus, besides having a mechanical function, tracheal cartilage also appears to be a local signaling structure in the embryonic lung. some pieces of this puzzle are still missing, and are currently under investigation. nevertheless, we have identified an important mechanism by which mesenchymal sox9-expressing cells instruct tracheal cartilage, epithelial, and lymphatic differentiation in a phenotype that already appears to model and may perhaps explain mechanistically some extreme cases of primary tracheomalacia in human new born infants. the appropriate temporospatial expression of sox in lung mesenchyme is necessary for correct tracheal cartilage formation, lymphatic vasculature system development, and epithelial differentiation. our study showed that a lack of tracheal cartilage rings resulted in increase in clara cells and decrease in basal and goblet cells in the tracheal epithelium. we also uncovered a novel mechanism of lung epithelium differentiation; tracheal cartilage rings instruct the tracheal epithelium to differentiate properly during embryonic development. thus, besides having a mechanical function, tracheal cartilage also appears to be a local signaling structure in the embryonic lung. 
sox is expressed in both distal lung epithelium and proximal lung mesenchyme. embryonic lung development is instructed by crosstalk between mesenchyme and epithelia, which results in activation of transcriptional factors, such as sox in a temporospatial manner. here, we investigated the effect of lung mesenchyme-specific inducible deletion of sox during murine lung development. the appropriate temporospatial expression of sox in lung mesenchyme is necessary for appropriate tracheal cartilage formation, lymphatic vasculature system development, and epithelial differentiation. cartilage rings were missing, and the tracheal lumen was collapsed in the mutant trachea. we uncovered a novel mechanism of lung epithelium differentiation: tracheal cartilage rings instruct the tracheal epithelium to differentiate properly during embryonic development. we also found that compared with normal tracheal epithelium, the mutant tracheal epithelium had an altered morphology with fewer p63-positive cells and more cc10-positive cells, fewer goblet cells, and downregulation of surfactant proteins a and c. in situ hybridization showed an altered expression pattern of tbx tbx and fgf genes and marked reduction of collagen expression in the tracheal mesenchyme. thus, besides having a mechanical function, tracheal cartilage also appears to be a local signaling structure in the embryonic lung. the tracheal phenotype was increasingly severe, with longer duration of deletion. transgenic mice lacking sox expression were unable to breathe and died at birth, with noticeable tracheal defects. lymphatic vasculature was underdeveloped in the mutant trachea: prox lyve and vegfr were decreased after sox knockout.
 embryonic lung development is instructed by crosstalk between mesenchyme and epithelia, which results in activation of transcriptional factors, such as sox in a temporospatial manner. sox is expressed in both distal lung epithelium and proximal lung mesenchyme. here, we investigated the effect of lung mesenchyme-specific inducible deletion of sox during murine lung development.  transgenic mice lacking sox expression were unable to breathe and died at birth, with noticeable tracheal defects. cartilage rings were missing, and the tracheal lumen was collapsed in the mutant trachea. in situ hybridization showed an altered expression pattern of tbx tbx and fgf genes and marked reduction of collagen expression in the tracheal mesenchyme. the tracheal phenotype was increasingly severe, with longer duration of deletion. lymphatic vasculature was underdeveloped in the mutant trachea: prox lyve and vegfr were decreased after sox knockout. we also found that compared with normal tracheal epithelium, the mutant tracheal epithelium had an altered morphology with fewer p63-positive cells and more cc10-positive cells, fewer goblet cells, and downregulation of surfactant proteins a and c.  the appropriate temporospatial expression of sox in lung mesenchyme is necessary for appropriate tracheal cartilage formation, lymphatic vasculature system development, and epithelial differentiation. we uncovered a novel mechanism of lung epithelium differentiation: tracheal cartilage rings instruct the tracheal epithelium to differentiate properly during embryonic development. thus, besides having a mechanical function, tracheal cartilage also appears to be a local signaling structure in the embryonic lung. sox9trachealungcartilagecc10p63 