BACKGROUND
the mouse lung arises from the anterior endoderm and forms during five overlapping phases of lung development: embryonic   <dig> to  <dig> ), pseudoglandular , canalicular , saccular  5) and alveolar   <cit> .
at the end of the embryonic phase, primary and secondary lung buds formation has taken place and the embryonic lung consists of one left lobe and four right lobes.
from e <dig>  to e <dig> , the epithelium undergoes branching morphogenesis to form the respiratory  tree.
in parallel to branching morphogenesis, the airway epithelium differentiates from a morphologically uniform cell population to different specialized cell types, thereby establishing a proximal-distal axis in the developing lung.
however, most of the differentiation occurs in the canalicular and saccular phases .
the primitive lung epithelium co-expresses several lineage markers including clara cell-specific 10 kda protein  and surfactant-associated protein c .
later in gestation , scgb1a <dig> is a marker for the proximal epithelium, whereas sftpc expression defines the distal epithelium.
in the adult lung these markers are characteristic of distinct cell lineages, scgb1a <dig> of clara cells and sftpc of alveolar type ii cells.
only specific progenitor cells in the adult lung, bronchioalveolar stem cells , co-express scgb1a <dig> and sftpc <cit> .
several evolutionarily conserved signaling pathways have been implicated in different phases of embryonic lung development.
in particular, members of the fibroblast growth factor, bone morphogenetic protein, hedgehog/gli, epidermal growth factor and wingless secreted glycoprotein  families have been implicated in lung morphogenesis and epithelial differentiation  <cit> .
in addition, a well-organized and balanced interplay between these signaling pathways and key transcription factors of lung development, including nk <dig> homeobox  <dig> , forkhead box protein a <dig>  and gata <dig>  is required for proper lung formation  <cit> .
gata <dig> is the only member of the gata family of zinc finger transcription factors that is expressed in the distal epithelium of the developing lung  <cit> .
gata <dig> is essential for branching morphogenesis and regulates differentiation of distal lung epithelium  <cit> .
moreover, gata <dig> has been implicated in blocking wnt signaling to control the balance between basc expansion and lung epithelial differentiation required for both lung development and regeneration  <cit> .
high mobility group at-hook protein  <dig>  is a transcription regulator belonging to the family of hmg proteins.
hmg proteins are the most abundant non-histone chromatin-associated proteins and regulate gene expression by altering chromatin structure and recruiting other proteins to the transcription regulatory complex  <cit> .
hmga <dig> is present at high levels in various undifferentiated tissues during embryonic development and its levels are strongly reduced in the corresponding adult tissues  <cit> .
in addition, hmga <dig> expression in adult organs has been implicated in maintaining and activating stem/progenitor cells in different tissues  <cit> .
here, we show that hmga <dig> mrna levels are high during early stages of lung development, in which cells are undifferentiated, and become reduced and restricted to the distal airways as lung development progresses, coincident with cell differentiation.
analysis of the lung of hmga2-knockout  mice  <cit>  revealed enhanced canonical wnt signaling that led to increased cell proliferation, increased number of progenitor cells and reduced differentiation of the distal airway epithelium.
using a lung explants culture system, we confirmed the causal involvement of wnt signaling mediating the effect of hmga2-loss-of-function  and showed that hmga <dig> is required for proper branching morphogenesis during the formation of the bronchial tree.
furthermore, we showed that hmga <dig> regulates canonical wnt signaling at different points of the pathway.
increased expression of the secreted wnt glycoproteins might explain a paracrine effect by which hmga2-ko enhanced cell proliferation in the mesenchyme of the developing lung.
in addition, hmga2-mediated direct regulation of gata <dig> is crucial for fine-tuning the activity of wnt signaling in the airway epithelium.
RESULTS
hmga <dig> is expressed in the mouse embryonic lung at the distal airways
to verify that hmga <dig> is expressed during lung development, quantitative reverse transcription pcr  expression analysis was performed .
hmga <dig> transcript was detected at e <dig> , when the primary lung buds have evaginated from the foregut and secondary buds are forming as outgrowths from the primary lung buds.
hmga <dig> expression progressively decreased during the pseudoglandular stages of lung development .
between the canalicular  and initial saccular stages , the levels of hmga <dig> transcript increased again.
later in gestation , hmga <dig> expression was further reduced and reached a basal level of expression that was maintained through the alveolar phase .
thus, hmga <dig> mrna levels were high during early stages of lung development, in which cells are undifferentiated, and decreased as lung development progressed, coincident with cell differentiation.
in situ hybridization expression pattern analysis in the embryonic lung at e <dig>  , when branching morphogenesis of the lung bud is proceeding rapidly to establish the future bronchial tree, revealed that hmga <dig> is ubiquitously expressed with higher levels of expression at the tips of the growing lung buds.
interestingly, hmga <dig> expression became restricted to the distal lung endoderm at e <dig> .
consistently, immunostaining on sections of the embryonic lung at e <dig>   supported the presence of hmga <dig> in cells of the distal lung endoderm.
co-staining with an antibody specific for the nuclear envelope protein lamin b <dig>  demonstrated the nuclear localization of hmga <dig>  the observed expression patterns in embryonic lung suggest a role for hmga <dig> in epithelial differentiation.
hmga <dig> is required for proper differentiation of the distal epithelium during lung development
to determine the role of hmga <dig> during lung development, we analyzed the embryonic lung of hmga2-deficient mice   <cit> .
at e <dig> , when the bronchial tree is complete and the lung tissues are differentiating into different cell types that will constitute the lung after birth, hmga2-ko resulted in a reduced body weight and lung-to-body-wet-weight ratio .
macroscopically, the embryonic lung of hmga2-ko mice at e <dig>  had four lobes on the right side and one lobe on the left, indicating that the earliest events during lung development, specification of pulmonary endoderm and primary branching morphogenesis, occurred normally and do not require hmga <dig>  however, histological analysis of the embryonic lung at this stage  revealed a marked increase of cell density in the hmga2
−/−
mice when compared with the wild-type  mice, suggesting increased cell proliferation.
furthermore, immunostaining on sections of embryonic lung  using antibodies specific for the epithelial marker pan-cytokeratin  and the mesenchymal marker vimentin  showed a broader mesenchyme and an irregularly shaped epithelium in hmga2-ko mice when compared to the wt mice.
this suggests an expansion of the mesenchyme at the expense of the epithelium.
consistent with this observation, quantification of krt-positive cells  showed a decrease from  <dig> % to  <dig> % , whereas the number of vim-positive cells  increased from  <dig> % to  <dig> %  in hmga2-ko mice when compared to the wt mice.
immunostaining on sections of embryonic lung showed increased levels of proliferation markers, specifically proliferating cell nuclear antigen  and antigen identified by monoclonal antibody ki  <dig>  , in both the epithelium and the mesenchyme of the hmga2-ko mice, as shown by co-staining with antibodies specific for krt  or vim , supporting our interpretation of the histological analysis.
however, quantification of our results  showed more prominent cell proliferation in the mesenchyme , from  <dig> % to  <dig> %  when compared to the epithelium , from 8% to  <dig> % .
to confirm increased cell proliferation in the embryonic lung after hmga2-ko, we analyzed the expression of cell-cycle progression markers cyclin e <dig>  e <dig> and d <dig> .
expression of these genes increased after hmga2-ko when compared to wt mice, demonstrating elevated cell proliferation.
in addition to cell proliferation, programmed cell death is an important process involved in lung development  <cit> .
thus, we performed co-staining on sections of embryonic lung  using antibodies specific for the active form of the apoptosis-related cysteine peptidase, cleaved caspase  <dig>  and krt  or the mesenchymal marker smooth muscle actin alpha  <dig> .
quantification of clcasp3-positive cells  showed that whereas the level of apoptosis in the mesenchyme was not significantly affected after hmga2-ko , it increased in the lung epithelium of hmga2
−/−
mice from  <dig> % to  <dig> % , supporting the reduction of epithelium when compared with wt mice.
however, since this increase of apoptosis cannot account for the drastic reduction of lung epithelium after hmga2-ko, we examined lung epithelium differentiation by analyzing the expression of proximal, scgb1a <dig>  and distal, sftpc, epithelial markers in the embryonic lung of wt and hmga2-ko mice .
whereas scgb1a <dig> expression did not change significantly, sftpc expression was reduced after hmga2-ko, suggesting defects in distal epithelium differentiation.
these defects were confirmed by immunostaining on sections of embryonic lung  that showed reduced numbers of sftpc-positive cells from  <dig> % to  <dig> %  after hmga2-ko when compared with the wt mice.
these results were confirmed in lung explants after sirna-mediated hmga <dig> knockdown  .
to further investigate these results, we performed immunostaining on sections of embryonic lung using antibodies specific for the distal epithelium progenitor cell marker sex determining region y-box  <dig>  and pcna .
the number of sox9-positive cells increased from  <dig> % to  <dig> %  after hmga2-ko when compared with wt mice.
interestingly, most of the sox9-positive cells in the hmga2-ko lung were proliferating as shown by pcna co-staining.
to summarize, hmga2-ko increased cell proliferation more significantly in the mesenchyme of the embryonic lung, increased apoptosis in the epithelium, and reduced differentiation of the distal airway epithelium by altering the balance between progenitor cell renewal and differentiation.
taking together, our data support that hmga <dig> is required for proper distal epithelium differentiation.
hmga <dig> knockout led to enhanced canonical wnt signaling
comparison of hmga2−/− with hmga2
+/+
mice by affymetrix microarray-based expression analysis of embryonic lung  revealed an increased expression of cell-cycle-related genes, confirming our immunohistological results.
we found that transcripts of genes that are either targets or positive regulators of canonical wnt signaling pathway increased in the embryonic lung of hmga2-ko mice.
furthermore, expression of negative regulators of canonical wnt signaling was reduced in hmga2-ko mice.
these results indicate enhancement of canonical wnt signaling after hmga2-ko that was further validated by increased activity of the beta-catenin/t-cell factor /lymphoid enhancer factor  wnt-reporter  in embryonic lung of the bat-gal:hmga2
−/−
double transgenic mice when compared to bat-gal:hmga2
+/+
.
western blot analysis of embryonic lung protein extracts  showed increased levels of activated-beta-catenin  and phosphorylation of the wnt co-receptor low-density lipoprotein receptor-related protein  <dig>   <cit>  after hmga2-ko, confirming enhancement of canonical wnt signaling.
however, beta-catenin  has a dual role in both wnt signaling and cell adhesion processes.
to confirm enhanced wnt signaling after hmga2-ko, we analyzed the expression of canonical wnt targets, axin <dig>  mycn , fgfr <dig>  and bmp <dig>  in the embryonic lung .
expression of canonical wnt targets increased after hmga2-ko when compared to wt mice, demonstrating elevated canonical wnt signaling.
western blot analysis of protein extracts from embryonic lung  showed increased levels of canonical wnt targets after hmga2-ko, confirming our expression analysis.
our results in the embryonic lung of hmga2
−/−
mice were supported by sirna-mediated hmga <dig> depletion in the mouse lung epithelial cell line mle- <dig> , which increased the basal transcription of a co-transfected wnt-responsive luciferase reporter  more than twofold when compared to control sirna -transfected cells.
interestingly, forced expression of hmga <dig> reduced the basal transcription of p3lef-luc, thereby supporting an hmga2-mediated negative regulation of wnt signaling.
enhanced canonical wnt signaling is related to cell proliferation  <cit> , correlating with our histological and molecular characterization of the hmga2-ko embryonic lung.
nevertheless, we observed increased cell proliferation in the mesenchyme of embryonic lung after hmga2-ko, although hmga <dig> expression is restricted to the distal epithelium of the embryonic lung.
a plausible explanation for these two observations could be that enhanced wnt signaling after hmga2-ko is induced in part by diffusible positive regulators of canonical wnt signaling.
indeed, our affymetrix microarray-based expression analysis  showed elevated expression of wnt <dig>  wnt7b and wnt2b in embryonic lung of hmga2
−/−
mice when compared to hmga2
+/+
.
these results were confirmed by qrt-pcr-based expression analysis .
furthermore, immunostaining on sections of embryonic lung using ctnnb1-specific antibody  showed increased translocation of ctnnb <dig> from the cytoplasm into the nucleus in cells of both the epithelium and the mesenchyme after hmga2-ko, demonstrating elevated canonical wnt signaling in both tissues and explaining hyperproliferation and expansion of the mesenchyme after hmga2-ko.
to confirm the causal involvement of canonical wnt signaling in mediating the effect of hmga2-lof, we used an organ explant culture system that mimics the normal embryonic lung development   <cit> .
depletion of hmga <dig> in these organ explants by sirna treatment  led to a marked impairment in branching morphogenesis when compared to explants exposed to control sirna  .
these defects during the formation of the bronchial and respiratory tree after hmga2-kd were validated by quantification of the total number of terminal branches and branch length .
when e <dig>  lungs were cultured for 72 hours in the presence of a hmga2-specific sirna , the total number of terminal branches was reduced from  <dig>  to  <dig> , and the branch length from 17 mm  to 12 mm .
however, treatment of the explants with the secreted canonical wnt inhibitor dickkopf homolog  <dig>   <cit>  antagonized the defects caused by sihmga <dig> , reconstituting the total number of terminal branches to  <dig>  and the branch length to 14 mm .
dkk <dig> treatment alone also affected branching morphogenesis by blocking wnt signaling as previously described from experiments in embryonic lung explants  <cit> .
western blot analysis of protein extracts from embryonic lung explants showed increased levels of abc and phosphorylated lrp <dig> after hmga2-kd , demonstrating enhanced canonical wnt signaling that was antagonized after dkk <dig> treatment .
the partial rescue of the hmga2-lof phenotype achieved by dkk1-mediated block of wnt signaling was validated by expression analysis of canonical wnt targets  and by western blot analysis of protein extracts from embryonic lung explants .
our data demonstrated the causal involvement of increased wnt signaling in mediating the effect of hmga2-lof, suggesting a role of hmga <dig> in negative regulation of wnt signaling that was supported by reduced activity of the wnt-responsive p3lef-luc reporter after hmga <dig> over-expression in mle- <dig> cells .
hmga <dig> directly activates gata <dig> expression
gata <dig> is a transcription factor that is essential for branching morphogenesis and inhibits canonical wnt signaling in the distal epithelium of the lung by transcriptional activation of its downstream target, the canonical wnt-beta-catenin pathway antagonist fzd <dig> <cit> .
the defects observed after hmga2-lof were similar to the gata6-lof phenotype   <cit> .
therefore, we investigated whether the enhancement of canonical wnt signaling after hmga2-lof could involve gata <dig> and fzd <dig>  immunostaining on sections of embryonic lung  using hmga2- and gata6-specific antibodies  showed the presence of both proteins in the same cells of the distal lung endoderm.
interestingly, expression analysis in embryonic lung showed that gata6- and fzd2-transcripts decreased after hmga2-ko when compared to wt mice .
consistently, sirna-mediated hmga2-lof reduced gata <dig> and fzd <dig> expression in embryonic lung explants .
western blot analysis of protein extracts from embryonic lung  showed reduced levels of gata <dig> and fzd <dig> after hmga2-ko, supporting our expression analysis.
to investigate the effect of hmga <dig> gain-of-function  on gata <dig> expression, mle- <dig> cells were transiently transfected with hmga <dig> and a plasmid containing the luciferase  reporter gene under the control of the gata <dig> promoter .
forced expression of hmga <dig> increased more than fivefold the basal transcription of the gata6-luc reporter.
in addition, expression of endogenous gata <dig> increased more than twofold after hmga <dig> transfection .
consistently, in silico analysis of the murine gata <dig> gene  revealed several hmga <dig> binding elements near the transcription initiation site.
moreover, chromatin immunoprecipitation assay  in mle- <dig> cells  showed a direct association of endogenous hmga <dig> to the endogenous gata <dig> promoter.
thus, we conclude that hmga <dig> directly activates gata <dig> gene expression, and the enhanced wnt signaling caused by hmga2-lof could also be mediated by a reduction of the wnt signaling antagonizing proteins gata <dig> and fzd <dig>
to show the requirement of gata <dig> in hmga2-mediated regulation of wnt signaling, we transfected mle- <dig> cells with hmga <dig> after sirna-mediated gata6- or fzd2-depletion .
expression analysis showed that hmga2-gof increased the levels of gata <dig> and fzd <dig> but reduced the levels of canonical wnt targets mycn, axin <dig> and bmp <dig>  confirming a role of hmga <dig> in inhibition of canonical wnt signaling.
interestingly, gata6- or fzd2-depletion antagonized the effect of hmga2-gof on both groups of markers analyzed, indicating the requirement of gata <dig> and fzd <dig> for the negative regulation of wnt signaling mediated by hmga <dig>
to determine the causal involvement of gata <dig> in hmga2-mediated regulation of wnt signaling, we transfected mle- <dig> cells with gata <dig> after sirna-mediated hmga2- or fzd2-depletion .
expression analysis showed that hmga2- and fzd2-lof enhanced the expression of canonical wnt targets, supporting our previous expression analysis in the embryonic lung of hmga2-ko mice and as expected from wnt signaling antagonizing genes.
interestingly, gata <dig> transfection compensated the effect of hmga2-lof, but not of fzd2-lof.
our data indicate that gata <dig> acts downstream of hmga <dig> and upstream of fzd <dig> in negative regulation of wnt signaling .
discussion and
CONCLUSIONS
we showed that hmga <dig> is expressed in the embryonic mouse lung at the distal airways.
hmga <dig> mrna levels were high during early stages of lung development, in which cells are undifferentiated, and decreased as lung development progressed, coincident with cell differentiation.
interestingly, we detected a slight increase of hmga <dig> expression at e <dig>  that matches with the establishment of a bipotent progenitor cell population in the distal epithelium  <cit> .
our data correlate with previous reports where hmga <dig> was shown to be present at high levels in various undifferentiated tissues during embryonic development and in strongly reduced levels in the corresponding adult tissues  <cit> .
hmga2-ko induces a pygmy phenotype due to reduced expression of insulin-like growth factor  <dig> mrna binding protein  <dig>   <cit> .
prior to our study, the lung of hmga2
−/−
mice had not been analyzed.
detailed analysis of hmga2
−/−
mice showed that hmga <dig> is required for distal epithelium differentiation during embryonic lung development.
hmga2-ko led to enhanced canonical wnt signaling due to an increase of secreted wnt glycoproteins as well as a reduction of the wnt signaling antagonizing proteins gata <dig> and fzd <dig>  thereby supporting that hmga <dig> regulates wnt signaling at different points of the pathway .
the causal involvement of canonical wnt signaling in mediating the effect of hmga2-lof was demonstrated by the dkk1-induced rescue of hmga2-lof in embryonic lung explants .
hmga2-mediated regulation of gata <dig> seems to be a key process in fine-tuning the activity of canonical wnt signaling in airway epithelium.
the sequential order of events suggested in our model  in which hmga <dig> acts upstream of gata <dig> is strongly supported by the fact that hmga <dig> directly regulates gata <dig>  as well as by the gata6-mediated rescue experiments of hmga2-lof in mle- <dig> cells .
since hmga <dig> regulates the transcription of its target genes by modulating the chromatin structure and by recruiting other proteins to the transcription regulatory complex  <cit> , the scope of our future work will be to investigate in detail the mechanism of hmga2-mediated transcriptional regulation of the gata <dig> promoter.
hmga2-ko increased cell proliferation not only in the lung epithelium, where hmga <dig> is expressed, but also in the mesenchyme, suggesting a paracrine effect that could be explained by increased expression of the secreted components of wnt signaling.
since hmga <dig> is known to activate transcription, the increased expression of wnt2b, wnt7b and wnt <dig> after hmga2-ko suggest the participation of a transcription inhibitor that could block the expression of these secreted components of wnt signaling and whose expression could be regulated by hmga <dig> .
identification of this unknown mediator of hmga <dig> will be the scope of future studies.
interestingly, analysis of the wnt7b promoter showed that deletion of the region between - <dig>  bp and -829 bp relative to the second transcription start site significantly increased the basal transcription activity of a wnt7b-luciferase reporter  <cit> , suggesting that the binding element of a putative transcription inhibitor was deleted in this construct.
the phenotypes of hmga2- and gata6-lof in embryonic lung explants are very similar   <cit> .
however, the milder phenotype observed in the embryonic lung of hmga2-ko mice when compared either with the gata6-ko or the phenotype induced after hmga2-kd in embryonic lung explants might be explained by redundancy in the function between hmga <dig> and hmga <dig>  another member of the hmg protein family.
hmga <dig> transcript was reduced after hmga2-lof  but not affected in the hmga2-ko mice .
hmga <dig> might compensate the hmga2-ko, thereby avoiding lethality at early embryonic stages, as is the case after gata6-ko  <cit> , or soon after birth due to defects in the lung, as is the case after lung epithelium-specific ablation of gata <dig> <cit> .
in addition, the expansion of the mesenchyme in the embryonic lung after hmga2-ko and the apparent increase of epithelium in embryonic lung explants after hmga2-kd might be explained by the differences of both lof systems.
in the transgenic approach, hmga2-lof takes place soon after fertilization and affects lung development from the initial stages of lung bud formation; in the explant culture, the lof starts at e <dig> , thereby reducing the rather indirect effect on the mesenchyme and making the effect on the epithelium more dominant.
analysis of the lungs in inducible and conditional double transgenic mice  would test these hypotheses and should be the scope of future studies.
hmga <dig> expression is positively regulated by transforming growth factor beta  <dig> signaling  <cit> .
in addition, our data show that hmga <dig> antagonizes canonical wnt signaling.
therefore, it will be of interest to determine a potential opposing effect between these two signaling pathways in establishing the proximal-distal axis during branching morphogenesis and lung epithelium differentiation in the developing lung.
hmga <dig> might play a crucial role on the interplay between these signaling pathways.
organ regeneration requires a proper balance between self-renewal and differentiation of tissue-specific progenitor cells.
canonical wnt signaling has been implicated in different regenerative processes including zebrafish tail regeneration, zebrafish cardiac regeneration and expansion of anterior heart field progenitors in mammals  <cit> .
moreover, canonical wnt signaling is activated upon lung epithelial regeneration, and enhanced wnt activity caused by lung epithelium-specific ablation of gata <dig> led to a premature and increased number of bascs  <cit> .
bascs represent one of several regional progenitor cell populations in the adult lung and are responsible for regeneration of bronchiolar and alveolar epithelium during homeostatic turnover and in response to injury  <cit> .
our study suggests the possible role of hmga <dig> in the adult lung controlling the balance between basc expansion and differentiation.
our results are the starting point for future studies in which the relevance of hmga2-mediated regulation of wnt signaling might be investigated in the adult lung within the context of proper balance between differentiation and self-renewal of lung stem/progenitor cells and lung regeneration during both homeostatic turnover and repair after injury.
characterization of the regulatory mechanisms controlling the proper balance between expansion and differentiation of lung stem/progenitor cells will have a profound impact on our understanding and treatment of lung disease.

 the mouse lung arises from the anterior endoderm and forms during five overlapping phases of lung development: embryonic to ), pseudoglandular, canalicular, saccular 5) and alveolar. at the end of the embryonic phase, primary and secondary lung buds formation has taken place and the embryonic lung consists of one left lobe and four right lobes. from e to e, the epithelium undergoes branching morphogenesis to form the respiratory tree. in parallel to branching morphogenesis, the airway epithelium differentiates from a morphologically uniform cell population to different specialized cell types, thereby establishing a proximal-distal axis in the developing lung. however, most of the differentiation occurs in the canalicular and saccular phases. the primitive lung epithelium co-expresses several lineage markers including clara cell-specific 10 kda protein and surfactant-associated protein c. later in gestation, scgb1a is a marker for the proximal epithelium, whereas sftpc expression defines the distal epithelium. in the adult lung these markers are characteristic of distinct cell lineages, scgb1a of clara cells and sftpc of alveolar type ii cells. only specific progenitor cells in the adult lung, bronchioalveolar stem cells, co-express scgb1a and sftpc. several evolutionarily conserved signaling pathways have been implicated in different phases of embryonic lung development. in particular, members of the fibroblast growth factor, bone morphogenetic protein, hedgehog/gli, epidermal growth factor and wingless secreted glycoprotein families have been implicated in lung morphogenesis and epithelial differentiation. in addition, a well-organized and balanced interplay between these signaling pathways and key transcription factors of lung development, including nk homeobox, forkhead box protein a and gata is required for proper lung formation. gata is the only member of the gata family of zinc finger transcription factors that is expressed in the distal epithelium of the developing lung. gata is essential for branching morphogenesis and regulates differentiation of distal lung epithelium. moreover, gata has been implicated in blocking wnt signaling to control the balance between basc expansion and lung epithelial differentiation required for both lung development and regeneration. high mobility group at-hook protein is a transcription regulator belonging to the family of hmg proteins. hmg proteins are the most abundant non-histone chromatin-associated proteins and regulate gene expression by altering chromatin structure and recruiting other proteins to the transcription regulatory complex. hmga is present at high levels in various undifferentiated tissues during embryonic development and its levels are strongly reduced in the corresponding adult tissues. in addition, hmga expression in adult organs has been implicated in maintaining and activating stem/progenitor cells in different tissues. here, we show that hmga mrna levels are high during early stages of lung development, in which cells are undifferentiated, and become reduced and restricted to the distal airways as lung development progresses, coincident with cell differentiation. analysis of the lung of hmga2-knockout mice revealed enhanced canonical wnt signaling that led to increased cell proliferation, increased number of progenitor cells and reduced differentiation of the distal airway epithelium. using a lung explants culture system, we confirmed the causal involvement of wnt signaling mediating the effect of hmga2-loss-of-function and showed that hmga is required for proper branching morphogenesis during the formation of the bronchial tree. furthermore, we showed that hmga regulates canonical wnt signaling at different points of the pathway. increased expression of the secreted wnt glycoproteins might explain a paracrine effect by which hmga2-ko enhanced cell proliferation in the mesenchyme of the developing lung. in addition, hmga2-mediated direct regulation of gata is crucial for fine-tuning the activity of wnt signaling in the airway epithelium.  hmga is expressed in the mouse embryonic lung at the distal airways to verify that hmga is expressed during lung development, quantitative reverse transcription pcr expression analysis was performed. hmga transcript was detected at e, when the primary lung buds have evaginated from the foregut and secondary buds are forming as outgrowths from the primary lung buds. hmga expression progressively decreased during the pseudoglandular stages of lung development. between the canalicular and initial saccular stages, the levels of hmga transcript increased again. later in gestation, hmga expression was further reduced and reached a basal level of expression that was maintained through the alveolar phase. thus, hmga mrna levels were high during early stages of lung development, in which cells are undifferentiated, and decreased as lung development progressed, coincident with cell differentiation. in situ hybridization expression pattern analysis in the embryonic lung at e, when branching morphogenesis of the lung bud is proceeding rapidly to establish the future bronchial tree, revealed that hmga is ubiquitously expressed with higher levels of expression at the tips of the growing lung buds. interestingly, hmga expression became restricted to the distal lung endoderm at e. consistently, immunostaining on sections of the embryonic lung at e supported the presence of hmga in cells of the distal lung endoderm. co-staining with an antibody specific for the nuclear envelope protein lamin b demonstrated the nuclear localization of hmga the observed expression patterns in embryonic lung suggest a role for hmga in epithelial differentiation. hmga is required for proper differentiation of the distal epithelium during lung development to determine the role of hmga during lung development, we analyzed the embryonic lung of hmga2-deficient mice. at e, when the bronchial tree is complete and the lung tissues are differentiating into different cell types that will constitute the lung after birth, hmga2-ko resulted in a reduced body weight and lung-to-body-wet-weight ratio. macroscopically, the embryonic lung of hmga2-ko mice at e had four lobes on the right side and one lobe on the left, indicating that the earliest events during lung development, specification of pulmonary endoderm and primary branching morphogenesis, occurred normally and do not require hmga however, histological analysis of the embryonic lung at this stage revealed a marked increase of cell density in the hmga2 −/− mice when compared with the wild-type mice, suggesting increased cell proliferation. furthermore, immunostaining on sections of embryonic lung using antibodies specific for the epithelial marker pan-cytokeratin and the mesenchymal marker vimentin showed a broader mesenchyme and an irregularly shaped epithelium in hmga2-ko mice when compared to the wt mice. this suggests an expansion of the mesenchyme at the expense of the epithelium. consistent with this observation, quantification of krt-positive cells showed a decrease from % to %, whereas the number of vim-positive cells increased from % to % in hmga2-ko mice when compared to the wt mice. immunostaining on sections of embryonic lung showed increased levels of proliferation markers, specifically proliferating cell nuclear antigen and antigen identified by monoclonal antibody ki, in both the epithelium and the mesenchyme of the hmga2-ko mice, as shown by co-staining with antibodies specific for krt or vim, supporting our interpretation of the histological analysis. however, quantification of our results showed more prominent cell proliferation in the mesenchyme, from % to % when compared to the epithelium, from 8% to %. to confirm increased cell proliferation in the embryonic lung after hmga2-ko, we analyzed the expression of cell-cycle progression markers cyclin e e and d. expression of these genes increased after hmga2-ko when compared to wt mice, demonstrating elevated cell proliferation. in addition to cell proliferation, programmed cell death is an important process involved in lung development. thus, we performed co-staining on sections of embryonic lung using antibodies specific for the active form of the apoptosis-related cysteine peptidase, cleaved caspase and krt or the mesenchymal marker smooth muscle actin alpha. quantification of clcasp3-positive cells showed that whereas the level of apoptosis in the mesenchyme was not significantly affected after hmga2-ko, it increased in the lung epithelium of hmga2 −/− mice from % to %, supporting the reduction of epithelium when compared with wt mice. however, since this increase of apoptosis cannot account for the drastic reduction of lung epithelium after hmga2-ko, we examined lung epithelium differentiation by analyzing the expression of proximal, scgb1a and distal, sftpc, epithelial markers in the embryonic lung of wt and hmga2-ko mice. whereas scgb1a expression did not change significantly, sftpc expression was reduced after hmga2-ko, suggesting defects in distal epithelium differentiation. these defects were confirmed by immunostaining on sections of embryonic lung that showed reduced numbers of sftpc-positive cells from % to % after hmga2-ko when compared with the wt mice. these results were confirmed in lung explants after sirna-mediated hmga knockdown. to further investigate these results, we performed immunostaining on sections of embryonic lung using antibodies specific for the distal epithelium progenitor cell marker sex determining region y-box and pcna. the number of sox9-positive cells increased from % to % after hmga2-ko when compared with wt mice. interestingly, most of the sox9-positive cells in the hmga2-ko lung were proliferating as shown by pcna co-staining. to summarize, hmga2-ko increased cell proliferation more significantly in the mesenchyme of the embryonic lung, increased apoptosis in the epithelium, and reduced differentiation of the distal airway epithelium by altering the balance between progenitor cell renewal and differentiation. taking together, our data support that hmga is required for proper distal epithelium differentiation. hmga knockout led to enhanced canonical wnt signaling comparison of hmga2−/− with hmga2 +/+ mice by affymetrix microarray-based expression analysis of embryonic lung revealed an increased expression of cell-cycle-related genes, confirming our immunohistological results. we found that transcripts of genes that are either targets or positive regulators of canonical wnt signaling pathway increased in the embryonic lung of hmga2-ko mice. furthermore, expression of negative regulators of canonical wnt signaling was reduced in hmga2-ko mice. these results indicate enhancement of canonical wnt signaling after hmga2-ko that was further validated by increased activity of the beta-catenin/t-cell factor /lymphoid enhancer factor wnt-reporter in embryonic lung of the bat-gal:hmga2 −/− double transgenic mice when compared to bat-gal:hmga2 +/+. western blot analysis of embryonic lung protein extracts showed increased levels of activated-beta-catenin and phosphorylation of the wnt co-receptor low-density lipoprotein receptor-related protein after hmga2-ko, confirming enhancement of canonical wnt signaling. however, beta-catenin has a dual role in both wnt signaling and cell adhesion processes. to confirm enhanced wnt signaling after hmga2-ko, we analyzed the expression of canonical wnt targets, axin mycn, fgfr and bmp in the embryonic lung. expression of canonical wnt targets increased after hmga2-ko when compared to wt mice, demonstrating elevated canonical wnt signaling. western blot analysis of protein extracts from embryonic lung showed increased levels of canonical wnt targets after hmga2-ko, confirming our expression analysis. our results in the embryonic lung of hmga2 −/− mice were supported by sirna-mediated hmga depletion in the mouse lung epithelial cell line mle-, which increased the basal transcription of a co-transfected wnt-responsive luciferase reporter more than twofold when compared to control sirna -transfected cells. interestingly, forced expression of hmga reduced the basal transcription of p3lef-luc, thereby supporting an hmga2-mediated negative regulation of wnt signaling. enhanced canonical wnt signaling is related to cell proliferation, correlating with our histological and molecular characterization of the hmga2-ko embryonic lung. nevertheless, we observed increased cell proliferation in the mesenchyme of embryonic lung after hmga2-ko, although hmga expression is restricted to the distal epithelium of the embryonic lung. a plausible explanation for these two observations could be that enhanced wnt signaling after hmga2-ko is induced in part by diffusible positive regulators of canonical wnt signaling. indeed, our affymetrix microarray-based expression analysis showed elevated expression of wnt wnt7b and wnt2b in embryonic lung of hmga2 −/− mice when compared to hmga2 +/+. these results were confirmed by qrt-pcr-based expression analysis. furthermore, immunostaining on sections of embryonic lung using ctnnb1-specific antibody showed increased translocation of ctnnb from the cytoplasm into the nucleus in cells of both the epithelium and the mesenchyme after hmga2-ko, demonstrating elevated canonical wnt signaling in both tissues and explaining hyperproliferation and expansion of the mesenchyme after hmga2-ko. to confirm the causal involvement of canonical wnt signaling in mediating the effect of hmga2-lof, we used an organ explant culture system that mimics the normal embryonic lung development. depletion of hmga in these organ explants by sirna treatment led to a marked impairment in branching morphogenesis when compared to explants exposed to control sirna. these defects during the formation of the bronchial and respiratory tree after hmga2-kd were validated by quantification of the total number of terminal branches and branch length. when e lungs were cultured for 72 hours in the presence of a hmga2-specific sirna, the total number of terminal branches was reduced from to, and the branch length from 17 mm to 12 mm. however, treatment of the explants with the secreted canonical wnt inhibitor dickkopf homolog antagonized the defects caused by sihmga, reconstituting the total number of terminal branches to and the branch length to 14 mm. dkk treatment alone also affected branching morphogenesis by blocking wnt signaling as previously described from experiments in embryonic lung explants. western blot analysis of protein extracts from embryonic lung explants showed increased levels of abc and phosphorylated lrp after hmga2-kd, demonstrating enhanced canonical wnt signaling that was antagonized after dkk treatment. the partial rescue of the hmga2-lof phenotype achieved by dkk1-mediated block of wnt signaling was validated by expression analysis of canonical wnt targets and by western blot analysis of protein extracts from embryonic lung explants. our data demonstrated the causal involvement of increased wnt signaling in mediating the effect of hmga2-lof, suggesting a role of hmga in negative regulation of wnt signaling that was supported by reduced activity of the wnt-responsive p3lef-luc reporter after hmga over-expression in mle- cells. hmga directly activates gata expression gata is a transcription factor that is essential for branching morphogenesis and inhibits canonical wnt signaling in the distal epithelium of the lung by transcriptional activation of its downstream target, the canonical wnt-beta-catenin pathway antagonist fzd. the defects observed after hmga2-lof were similar to the gata6-lof phenotype. therefore, we investigated whether the enhancement of canonical wnt signaling after hmga2-lof could involve gata and fzd immunostaining on sections of embryonic lung using hmga2- and gata6-specific antibodies showed the presence of both proteins in the same cells of the distal lung endoderm. interestingly, expression analysis in embryonic lung showed that gata6- and fzd2-transcripts decreased after hmga2-ko when compared to wt mice. consistently, sirna-mediated hmga2-lof reduced gata and fzd expression in embryonic lung explants. western blot analysis of protein extracts from embryonic lung showed reduced levels of gata and fzd after hmga2-ko, supporting our expression analysis. to investigate the effect of hmga gain-of-function on gata expression, mle- cells were transiently transfected with hmga and a plasmid containing the luciferase reporter gene under the control of the gata promoter. forced expression of hmga increased more than fivefold the basal transcription of the gata6-luc reporter. in addition, expression of endogenous gata increased more than twofold after hmga transfection. consistently, in silico analysis of the murine gata gene revealed several hmga binding elements near the transcription initiation site. moreover, chromatin immunoprecipitation assay in mle- cells showed a direct association of endogenous hmga to the endogenous gata promoter. thus, we conclude that hmga directly activates gata gene expression, and the enhanced wnt signaling caused by hmga2-lof could also be mediated by a reduction of the wnt signaling antagonizing proteins gata and fzd to show the requirement of gata in hmga2-mediated regulation of wnt signaling, we transfected mle- cells with hmga after sirna-mediated gata6- or fzd2-depletion. expression analysis showed that hmga2-gof increased the levels of gata and fzd but reduced the levels of canonical wnt targets mycn, axin and bmp confirming a role of hmga in inhibition of canonical wnt signaling. interestingly, gata6- or fzd2-depletion antagonized the effect of hmga2-gof on both groups of markers analyzed, indicating the requirement of gata and fzd for the negative regulation of wnt signaling mediated by hmga to determine the causal involvement of gata in hmga2-mediated regulation of wnt signaling, we transfected mle- cells with gata after sirna-mediated hmga2- or fzd2-depletion. expression analysis showed that hmga2- and fzd2-lof enhanced the expression of canonical wnt targets, supporting our previous expression analysis in the embryonic lung of hmga2-ko mice and as expected from wnt signaling antagonizing genes. interestingly, gata transfection compensated the effect of hmga2-lof, but not of fzd2-lof. our data indicate that gata acts downstream of hmga and upstream of fzd in negative regulation of wnt signaling. discussion and  we showed that hmga is expressed in the embryonic mouse lung at the distal airways. hmga mrna levels were high during early stages of lung development, in which cells are undifferentiated, and decreased as lung development progressed, coincident with cell differentiation. interestingly, we detected a slight increase of hmga expression at e that matches with the establishment of a bipotent progenitor cell population in the distal epithelium. our data correlate with previous reports where hmga was shown to be present at high levels in various undifferentiated tissues during embryonic development and in strongly reduced levels in the corresponding adult tissues. hmga2-ko induces a pygmy phenotype due to reduced expression of insulin-like growth factor mrna binding protein. prior to our study, the lung of hmga2 −/− mice had not been analyzed. detailed analysis of hmga2 −/− mice showed that hmga is required for distal epithelium differentiation during embryonic lung development. hmga2-ko led to enhanced canonical wnt signaling due to an increase of secreted wnt glycoproteins as well as a reduction of the wnt signaling antagonizing proteins gata and fzd thereby supporting that hmga regulates wnt signaling at different points of the pathway. the causal involvement of canonical wnt signaling in mediating the effect of hmga2-lof was demonstrated by the dkk1-induced rescue of hmga2-lof in embryonic lung explants. hmga2-mediated regulation of gata seems to be a key process in fine-tuning the activity of canonical wnt signaling in airway epithelium. the sequential order of events suggested in our model in which hmga acts upstream of gata is strongly supported by the fact that hmga directly regulates gata as well as by the gata6-mediated rescue experiments of hmga2-lof in mle- cells. since hmga regulates the transcription of its target genes by modulating the chromatin structure and by recruiting other proteins to the transcription regulatory complex, the scope of our future work will be to investigate in detail the mechanism of hmga2-mediated transcriptional regulation of the gata promoter. hmga2-ko increased cell proliferation not only in the lung epithelium, where hmga is expressed, but also in the mesenchyme, suggesting a paracrine effect that could be explained by increased expression of the secreted components of wnt signaling. since hmga is known to activate transcription, the increased expression of wnt2b, wnt7b and wnt after hmga2-ko suggest the participation of a transcription inhibitor that could block the expression of these secreted components of wnt signaling and whose expression could be regulated by hmga. identification of this unknown mediator of hmga will be the scope of future studies. interestingly, analysis of the wnt7b promoter showed that deletion of the region between -  bp and -829 bp relative to the second transcription start site significantly increased the basal transcription activity of a wnt7b-luciferase reporter, suggesting that the binding element of a putative transcription inhibitor was deleted in this construct. the phenotypes of hmga2- and gata6-lof in embryonic lung explants are very similar. however, the milder phenotype observed in the embryonic lung of hmga2-ko mice when compared either with the gata6-ko or the phenotype induced after hmga2-kd in embryonic lung explants might be explained by redundancy in the function between hmga and hmga another member of the hmg protein family. hmga transcript was reduced after hmga2-lof but not affected in the hmga2-ko mice. hmga might compensate the hmga2-ko, thereby avoiding lethality at early embryonic stages, as is the case after gata6-ko, or soon after birth due to defects in the lung, as is the case after lung epithelium-specific ablation of gata. in addition, the expansion of the mesenchyme in the embryonic lung after hmga2-ko and the apparent increase of epithelium in embryonic lung explants after hmga2-kd might be explained by the differences of both lof systems. in the transgenic approach, hmga2-lof takes place soon after fertilization and affects lung development from the initial stages of lung bud formation; in the explant culture, the lof starts at e, thereby reducing the rather indirect effect on the mesenchyme and making the effect on the epithelium more dominant. analysis of the lungs in inducible and conditional double transgenic mice would test these hypotheses and should be the scope of future studies. hmga expression is positively regulated by transforming growth factor beta signaling. in addition, our data show that hmga antagonizes canonical wnt signaling. therefore, it will be of interest to determine a potential opposing effect between these two signaling pathways in establishing the proximal-distal axis during branching morphogenesis and lung epithelium differentiation in the developing lung. hmga might play a crucial role on the interplay between these signaling pathways. organ regeneration requires a proper balance between self-renewal and differentiation of tissue-specific progenitor cells. canonical wnt signaling has been implicated in different regenerative processes including zebrafish tail regeneration, zebrafish cardiac regeneration and expansion of anterior heart field progenitors in mammals. moreover, canonical wnt signaling is activated upon lung epithelial regeneration, and enhanced wnt activity caused by lung epithelium-specific ablation of gata led to a premature and increased number of bascs. bascs represent one of several regional progenitor cell populations in the adult lung and are responsible for regeneration of bronchiolar and alveolar epithelium during homeostatic turnover and in response to injury. our study suggests the possible role of hmga in the adult lung controlling the balance between basc expansion and differentiation. our results are the starting point for future studies in which the relevance of hmga2-mediated regulation of wnt signaling might be investigated in the adult lung within the context of proper balance between differentiation and self-renewal of lung stem/progenitor cells and lung regeneration during both homeostatic turnover and repair after injury. characterization of the regulatory mechanisms controlling the proper balance between expansion and differentiation of lung stem/progenitor cells will have a profound impact on our understanding and treatment of lung disease. 
we found that hmga is expressed in the mouse embryonic lung at the distal airways. our results support that hmga regulates canonical wnt signaling at different points of the pathway. here we deciphered the role of the high-mobility-group at-hook protein during lung development by analyzing the lung of hmga2-deficient mice. mice showed that hmga is required for proper cell proliferation and distal epithelium differentiation during embryonic lung development. our results are the starting point for future studies investigating the relevance of hmga2-mediated regulation of wnt signaling in the adult lung within the context of proper balance between differentiation and self-renewal of lung stem/progenitor cells during lung regeneration in both homeostatic turnover and repair after injury. hmga knockout led to enhanced canonical wnt signaling due to an increased expression of secreted wnt glycoproteins wnt2b, wnt7b and wnt as well as a reduction of the wnt signaling antagonizing proteins gata-binding protein and frizzled homolog analysis of sirna-mediated loss-of-function experiments in embryonic lung explant culture confirmed the role of hmga as a key regulator of distal lung epithelium differentiation and supported the causal involvement of enhanced canonical wnt signaling in mediating the effect of hmga2-loss-of-fuction. increased expression of the secreted wnt glycoproteins might explain a paracrine effect by which hmga2-knockout enhanced cell proliferation in the mesenchyme of the developing lung. the high-mobility-group proteins are the most abundant non-histone chromatin-associated proteins. hmg proteins are present at high levels in various undifferentiated tissues during embryonic development and their levels are strongly reduced in the corresponding adult tissues, where they have been implicated in maintaining and activating stem/progenitor cells. in addition, hmga2-mediated direct regulation of gata is crucial for fine-tuning the activity of wnt signaling in the airway epithelium.
 the high-mobility-group proteins are the most abundant non-histone chromatin-associated proteins. hmg proteins are present at high levels in various undifferentiated tissues during embryonic development and their levels are strongly reduced in the corresponding adult tissues, where they have been implicated in maintaining and activating stem/progenitor cells. here we deciphered the role of the high-mobility-group at-hook protein during lung development by analyzing the lung of hmga2-deficient mice.  we found that hmga is expressed in the mouse embryonic lung at the distal airways. analysis of hmga2 −/− mice showed that hmga is required for proper cell proliferation and distal epithelium differentiation during embryonic lung development. hmga knockout led to enhanced canonical wnt signaling due to an increased expression of secreted wnt glycoproteins wnt2b, wnt7b and wnt as well as a reduction of the wnt signaling antagonizing proteins gata-binding protein and frizzled homolog analysis of sirna-mediated loss-of-function experiments in embryonic lung explant culture confirmed the role of hmga as a key regulator of distal lung epithelium differentiation and supported the causal involvement of enhanced canonical wnt signaling in mediating the effect of hmga2-loss-of-fuction. finally, we found that hmga directly regulates gata and thereby modulates fzd expression.  our results support that hmga regulates canonical wnt signaling at different points of the pathway. increased expression of the secreted wnt glycoproteins might explain a paracrine effect by which hmga2-knockout enhanced cell proliferation in the mesenchyme of the developing lung. in addition, hmga2-mediated direct regulation of gata is crucial for fine-tuning the activity of wnt signaling in the airway epithelium. our results are the starting point for future studies investigating the relevance of hmga2-mediated regulation of wnt signaling in the adult lung within the context of proper balance between differentiation and self-renewal of lung stem/progenitor cells during lung regeneration in both homeostatic turnover and repair after injury. branching morphogenesishmga2gata6lung developmentwnt signaling 