BACKGROUND
the lung forms as two evaginations from the ventral foregut at e <dig> , a few days after the initial anterior to posterior specification of the uniform gut tube takes place.
sequential branching of the epithelium forms an intricate tree of airways with a distinct axis of proximal to distal differentiation, and a coordinated formation of blood vessels at the distal end.
an understanding of the budding process that leads to epithelial branching is quite advanced.
organ culture experiments have shown that branching morphogenesis depends on the presence of lung mesenchyme, which induces branching in tracheal epithelium  <cit> , and that the mesenchymal-expressed fibroblast growth factor  <dig>  can substitute for mesenchyme  <cit> .
the importance of fgf <dig> for lung development is demonstrated by the fact that fgf <dig> null mice die at birth due to numerous defects, one of them being the absence of lung buds  <cit> .
using lung explant culture bellusci et al and park et al demonstrated that fgf <dig> acts as a chemoattractant for the epithelium in lung buds in vitro  <cit> .
fgf <dig> expression studies suggest that fgf10-signaling plays an iterative role during lung branching morphogenesis in vivo, as fgf <dig> is expressed in a dynamic pattern at the tip of each forming bud  <cit> .
although it is still unknown precisely how this fgf <dig> expression pattern is controlled, factors that regulate fgf <dig> expression in the lung include fgf <dig>  <cit> , tgf-beta  <cit> , shh  <cit>  and bmp <dig>  <cit> , and interplay between the budding epithelium  and the mesenchyme causes a rapid downregulation of fgf <dig> as soon as budding is initiated.
in vivo studies of the mechanism by which fgf <dig> regulates development of the lung are limited, but a study employing transgenic overexpression of fgf <dig> by promoter elements from clara cell secretory protein  or surfactant protein c  genes demonstrated a pronounced prenatal and postnatal effect of fgf <dig> overexpression.
transgenic expression could be detected starting from e <dig>  after embryonic induction and resulted in perturbed branching morphogenesis and formation of adenomatous malformations in e <dig>  lungs, demonstrating that localized dynamic expression is the key to proper branching.
postnatal overexpression of fgf <dig> led to tumor formation and differentiation of epithelial tumor cells into a distal type ii alveolar cell like phenotype.
affected areas regressed into proximal cell types upon fgf <dig> cessation indicating that the differentiated distal cells in this transgenic setting required ongoing fgf <dig> signaling to be maintained  <cit> .
this result indicates that fgf <dig> may play a role in selectively regulating differentiation of the two different pulmonary cell lineages; the proximal conducting airways  and the distal respiratory airways .
the bronchial epithelium contains clara cells, ciliated cells and neuroendocrine cells, while the alveolar epithelium consists of type i and type ii alveolar cells.
since most of these cell types are specified already before birth, and ectopic fgf <dig> has the potential to balance cell types in favor of distal cells postnatally, it is possible that fgf <dig> plays a role in this prenatal specification, although this question has not yet been addressed.
fgf <dig> has been shown to regulate prenatal differentiation in other endodermal organs and act as a progenitor maintenance factor.
in the pancreas, fgf <dig> null mice are depleted of progenitor cells leading to arrest of growth and differentiation of the epithelium  <cit> , while overexpression causes expansion of progenitor cells and attenuation of differentiation  <cit> .
a similar function is seen in the gastrointestinal tract, as fgf <dig> null mice exhibit a decrease in dividing progenitor cells  <cit> , and ectopic overexpression leads to attenuation of differentiation in a lineage specific manner  <cit> .
the apparent dichotomy of an established role of fgf <dig> as a chemoattractant in the lung versus a progenitor maintenance factor in the gastrointestinal organs might be rooted in lack of available information due to the dramatic early phenotype in the lungs of fgf <dig> null mice.
indeed, a recent paper has demonstrated that dosage of fgf <dig> in the lungs is critical for progenitor cell amplification.
hypomorphic lungs exhibit severe hypoplasia most likely due to insufficient progenitor cell maintenance and reduced vascular development  <cit> .
during the course of our analyses of the role of fgf <dig> on more posterior endoderm , based on a model of ectopic expression of fgf <dig> using the supposedly pancreas/duodenal/gastric specific pdx <dig>  promoter element, we surprisingly detected a dramatic effect on lung development.
the transgenic model allowed us to address the hypothesis that fgf <dig> controls differentiation in the embryonic lung epithelium and functions in lung progenitor maintenance.
we here demonstrate that the endogenous pdx <dig> protein is expressed in developing lung at reduced levels compared to the previously described expression in posterior stomach, pancreas and anterior duodenum  <cit> , and we find elevated level of fgf <dig> mrna and protein in lungs of the ppdx1-fgf <dig> model from e <dig> .
we observe effects on multiple aspects of lung development upon ectopic presence of fgf <dig> within the developing lung epithelium: our studies confirm that epithelial branching is impaired in fgf <dig> overexpressing lung lobes  <cit> .
additional effects in the embryonic lung include increased cell proliferation, distalization of the epithelium, attenuation of terminal differentiation, and goblet cell metaplasia.
our work using a gain-of-function approach suggests that the tight control of fgf <dig> expression observed in the lung is important in controlling the balance between proliferation and differentiation as well as cell lineage determination.
RESULTS
fgf10flag is expressed in lung lobes of ppdx1-fgf10flag mice
transient transgenic mice expressing a flag-tagged version of mouse fgf <dig> under the pdx <dig> promoter elements were obtained by oocyte injection as previously described  <cit> .
although this pdx <dig> promoter is thought to be specific to the pancreas, duodenum and posterior stomach, we found a profound phenotype in the lung in approximately half of the transient transgenic  mice, causing perinatal lethality.
initial assessment of ppdx1-fgf10flag embryos with malformed lungs revealed enlargement of one or more lung lobes, and an increase in the distance between terminal branches .
the phenotype varied between individual mice; in two of the transgenic mice analyzed by histochemistry the entire lung was aberrantly formed, while the other two embryos had abnormal formation of only one of the right lobes or the entire left lobe.
immunohistochemistry for fgf <dig>  using an antibody that only detects high amounts of protein, as well as an antibody for the flag-tag demonstrated that epithelial cells in these lung lobes contained transgenic fgf <dig>  while the normal lobes did not .
some cells displayed abundant fgf <dig> expression in the entire cytoplasm , suggesting that these cells synthesize fgf <dig>  while the majority of cells only stained for fgf <dig> in the cell membrane .
in order to validate the production of transgenic fgf <dig> in the lung itself, we performed in situ hybridization for fgf <dig> and low cycle multiplex  rt-pcr for fgf10flag mrna using g6pd- <dig> as a control.
mpx rt-pcr for the transgenic construct showed that fgf10flag mrna was detectable in the tg lung .
in situ hybridization with a short detection time showed fgf <dig> in the epithelium of the transgenic lung lobes of e <dig>  and e <dig>  embryos , confirming ectopic presence of fgf <dig>  the highest fgf <dig> mrna expression was confined to small clusters of cells in the e <dig>  embryos  and to isolated cells at e <dig>  .
variation in expression of fgf <dig> in pancreas and stomach due to mosaicism in these embryos has been reported before  <cit> , in all cases, fgf <dig> protein expression was widespread due to the secreted nature of the protein .
the observed phenotype and the expression of fgf10flag mrna and protein in the transgenic lung demonstrate that the pdx <dig> promoter construct must be active in this tissue.
this was surprising given the previously reported very accurate replication of endogenous pdx <dig> expression conferred by the construct in the pancreas, duodenum and posterior stomach  <cit> .
as the pdx <dig> promoter fragment used is limited to app.
<dig>  kb upstream regulatory sequence, lung expression may be explained by absence of regulatory elements involved in repression of the pdx <dig> promoter outside the distal foregut domain.
however, it was also possible that pdx <dig> is normally expressed in lung at a low level and would therefore not have been previously detected.
we tested this latter hypothesis by performing an analysis of pdx <dig> expression throughout embryonic development.
our results revealed that pdx <dig> protein is present in the early lung buds at e <dig> , albeit at a very low level compared to pancreatic and duodenal expression .
we were unable to detect any pdx <dig> expression in e <dig>   and e <dig>   wildtype lungs, but in transgene expressing lung lobes significant pdx <dig> protein expression was maintained in clusters of epithelial cells .
these lobes exhibited the transgenic phenotype in the entire lobe, likely attributed to the secreted nature of fgf <dig>  not all pdx <dig> expressing cells displayed high levels of cytoplasmic staining for fgf <dig>  but all fgf <dig> secreting cells were positive for pdx <dig> .
these observations argue that fgf <dig> is capable of autocrine maintenance of cells with pdx <dig> expression, otherwise lost during development.
disruption of branching morphogenesis and bronchiole formation
histological examination of transgenic e <dig>  lungs revealed that the increased size observed was due to hyperplastic growth as well as formation of large empty lumens surrounded by a mesenchymal layer .
branching morphogenesis was disrupted leading to formation of dense areas of tissue composed of mesenchyme and epithelium, somewhat resembling the pseudoglandular stage .
proximal bronchi were formed to some degree, but terminal bronchioles were absent .
these results support previous findings  <cit>  that fgf <dig> overexpression in the lung is lethal at birth due to pulmonary defects including perturbed branching morphogenesis.
in embryos with only one malformed lobe, the surrounding lobes appeared normal .
throughout this study we show data obtained from one embryo with a single malformed lobe  as well as data obtained from one transgenic embryo with the entire lung malformed and one wildtype littermate embryo , as each was representative for their phenotype.
as we cannot exclude that wildtype-like lobes in the transgenic embryos have been exposed to ectopic fgf <dig> early in development, the tg lung lobes displaying wildtype morphology are labeled "wt" on the figures also containing tg lung lobes with a pronounced phenotype.
fgf <dig> overexpression results in lung hyperplasia due to increased cell proliferation
the number of cells undergoing mitosis at e <dig>  in the alveolar tissue is normally negligible , although the entire epithelium in the bronchi is still positive for proliferating cell nuclear antigen  .
in contrast, the majority of cells were pcna positive throughout the transgenic lung lobes, in mesenchyme and epithelium , demonstrating that transgenic lung lobes are still in a proliferative state.
indeed, as judged from phosphorylated histone h <dig>  staining, many cells were in m-phase in the tg lobe, but very few in the "wt" lobe .
these results support previous findings that fgf <dig> stimulates pulmonary proliferation in vitro  <cit>  and correspond with the reduction of epithelial cell proliferation seen in fgf <dig> hypomorphic mice  <cit> .
fgf <dig> promotes formation of smooth muscle
fgf <dig> is expressed in parabronchial smooth muscle progenitors and mice hypomorphic for fgf <dig> and mice with a fgfr2b splicing defect exhibit a reduction in smooth muscle cells  <cit> .
we therefore proceeded to analyze expression of smooth muscle actin by immunohistochemistry.
parabronchial smooth muscle cells seemed unaltered in those cases where proximal bronchi did form, but unlike wildtype lungs, transgenic lungs had developed a layer of smooth muscle actin positive cells surrounding the perimeter of the lung lobe .
a few cells within this band were actively dividing  .
attenuation of alveolar differentiation and retention of markers of the undifferentiated distal buds
the high number of cells still undergoing mitosis at e <dig>  indicated that transgenic pulmonary cells had not differentiated to the same extent as wildtype.
pro-surfactant protein c is expressed as early as e <dig>  in the murine lung progenitor cells  <cit>  and is also expressed by alveolar type ii cells developing before birth.
this protein was expressed in both tg and wt lungs, but was expressed in cuboidal cells arranged in a simple epithelium in the transgenic lobes  as opposed to the rounded solitary alveolar type ii cells in wt lungs .
the majority of transgenic epithelial cells were pro-surfactant protein c positive.
analysis of expression of the receptor for advanced glycation end-products , a marker for alveolar type i cells   <cit>  revealed an almost complete absence in tg lungs .
type i alveolar cells are abundant in e <dig>  wildtype lungs and constitute a major part of the alveolar epithelium, with type ii alveolar cells occupying the corners between type i cells .
the absence of type i cells in the transgenic lobes and the presence of pro-surfactant protein c expressing cells that did not resemble mature type ii cells, suggested that differentiation was inhibited.
further characterization of the pro-surfactant protein c expressing cells showed that these cells were actively dividing , and that they expressed the ets factor pea <dig>  , which is a fgfr-signaling target and a marker of distal bud cells  <cit> .
this transcription factor was only expressed in a few cells in the wildtype alveolar tissue, and was not expressed in type ii cells .
in order to determine if fgf <dig> overexpression had resulted in arrest of pulmonary cells in a state resembling the undifferentiated distal bud cells, as suggested by pea expression, we analyzed the presence of several other markers that characterize these cells.
ttf <dig>  encodes a lung-specific homeodomain factor that is expressed from e <dig>  in the lung bud epithelium and after birth in type ii cells  <cit> .
we detected presence of ttf <dig> in transgenic epithelial cells, but only in a few peripheral wildtype cells .
clusterin is a glycoprotein that is associated with cell damage in adult cells and with differentiation during development.
it is expressed transiently during branching morphogenesis in the lung and is localized in the epithelial cells of the distal buds  <cit> .
clusterin was not expressed in wt e <dig>  lung, but was expressed in transgenic epithelial cells facing the lumen .
ttf <dig>  clusterin and pro-surfactant protein c were co-expressed by the transgenic epithelial cells , similar to what is seen in the distal tips during the late pseudoglandular stage  .
the observed expansion of the expression pattern of several early distal markers indicate that fgf <dig> overexpression leads to distalization of the entire embryonic lung, similar to what has previously been found in postnatal lung tumors  <cit> .
the absence of mature distal type i and type ii cells furthermore indicate that the transgenic cells, although specified to a distal fate, are arrested in a progenitor-like state.
bronchi and bronchiole formation and differentiation are disrupted by fgf10
initial assessment of the phenotype revealed that terminal bronchioles were missing, while proximal bronchi development had taken place to some degree, but had differentiation of the bronchial cell lineages taken place?
analysis for expression of uteroglobin, a clara cell marker, revealed that, as expected from the lack of terminal bronchiole development, there were no clara cells in the distal lung .
proximal bronchi development was also perturbed as the number of cells expressing uteroglobin was reduced compared to wildtype .
sox <dig> is expressed in ciliated cells of wildtype trachea and bronchi , and expression of this marker was greatly reduced by fgf <dig>  ciliated cells were found in the most proximal areas, but were similarly reduced as the clara cells , while they were almost absent from more distal areas .
only a few scattered ciliated cells remained in the distal epithelium suggesting that bronchiole development was aborted .
interestingly, one particular bronchial lineage descendant was capable of forming.
cgrp expressing pulmonary neuroendocrine cells  were still present in the transgenic lung lobes.
in the wildtype lung these cells were confined to bronchi and bronchioles and appeared in small clusters , while they were scattered throughout the transgenic lung .
our results support the assessment that bronchiole development was terminated, as only pnecs were present in the distal transgenic lung.
expression of pdx <dig> in the lung does not lead to transdifferentiation to intestinal, gastric or pancreatic fates
the observation that transgenic lungs lacked terminal pulmonary cells lead to speculations of whether transdifferentiation to a pancreatic, gastric or intestinal fate had taken place, due to ectopic expression of fgf <dig> and pdx <dig>  we performed immunohistochemistry for expression of nkx <dig>   and insulin, but did not detect expression, indicating that transdifferentiation to a pancreatic fate did not occur .
cdx <dig> is expressed throughout the intestine, math <dig> is expressed by intestinal secretory cell lineages, cryptdin <dig> by paneth cells and intestinal fatty acid binding protein  by enterocytes.
in the transgenic lungs we could not detect cdx <dig> or fabp <dig> expression , and only very few cryptdin <dig> or math <dig> expressing cells .
sox <dig> is expressed in the gastric epithelium, throughout the esophagus, and is present in the ciliated cells in the bronchi  <cit> , but very little expression was seen in the transgenic tissue outside of bronchi .
likewise, h/k-atpase and pepsinogenc, expressed by parietal and chief cells respectively, were also absent .
trefoil factor  <dig>  is normally expressed by mucous producing cells in the stomach , and is absent from embryonic lung .
a few tff <dig> positive cells were found in one out of three transgenic mice .
tff <dig> is expressed in intestinal goblet cells, and although not expressed in embryonic lung , is expressed by pulmonary goblet cells in the tracheal submucosal glands in adult mice  <cit> .
tff <dig> was expressed in several cells throughout the distal epithelium in all transgenic lobes .
we therefore conclude that transdifferentiation to a gut like fate had not taken place, although limited expression of math <dig> and tff <dig> as well as high expression of tff <dig> pointed towards a possible goblet cell metaplasia.
embryonic goblet cell metaplasia
goblet cells are present in only few numbers in submucosal glands of adult mice although more cells can form as a response to inflammation in adult mice and humans, and in some human lung cancers.
goblet cells react with dolichos biflorus agglutinin   <cit> , and detection of rhodamine or cy <dig> conjugated dba, as well as pas/alcian blue histochemistry , confirmed that goblet cells were present in the distal epithelium of transgenic but not wildtype lobes .
no dba reactivity was found in bronchi or trachea.
since this goblet cell metaplasia had arisen in the prospective alveolar epithelium, we performed a double immunofluorescence analysis for dba and pro-surfactant protein c. similar to wt lung, dba and p-sftpc were not co-expressed .
increased beta-catenin signaling and bmp <dig> expression
regulation of transcription through activation of stabilized beta-catenin is necessary for differentiation of distal airways  <cit> , and transgenic expression of an activated form of beta-catenin leads to goblet cell metaplasia and expansion of alveolar type ii-like cells  <cit> .
since goblet cell metaplasia and expansion of surfactant protein c expressing cells were also observed in the ppdx1-fgf10flag model, we next tested whether the observed phenotype could be caused partly through activation of beta-catenin .
we analyzed for activation of beta-catenin using an antibody specific for the dephosphorylated serine <dig> or threonine <dig>  which is an activated form  <cit> .
cells positive for activated beta-catenin were present throughout the epithelium and mesenchyme in both wildtype and transgenic lobes , and more activated cells were present in the distal part of the wildtype lung , than the proximal part .
the frequency of activated beta-catenin cells was higher in the transgenic lobes, especially in the proximal lung  but also in the distal lung , suggesting that fgf <dig> overexpression leads to activation of beta-catenin.
bmp <dig> is expressed in the distal-most epithelial cells in proximity to the fgf <dig> expressing mesenchyme, and bmp <dig> expression is upregulated in response to a fgf <dig> bead  <cit> .
we therefore analyzed whether bmp <dig> expression was increased in the ppdx1-fgf10flag lung.
in situ hybridization revealed that bmp <dig> was expressed in scattered epithelial cells at e <dig>  , while the number of bmp <dig> positive cells was increased in transgenic lungs .
this result was confirmed by mpx rt-pcr, where bmp <dig> but not bmp <dig> expression was highly upregulated in transgenic lungs .
these results confirm previous in vitro data and demonstrate that fgf <dig> overexpression leads to activation of beta-catenin and upregulation of epithelial bmp <dig> expression, suggesting that these are downstream effectors of fgf <dig> and could be involved in the observed phenotype.
this observation is in agreement with previous reports showing that reductions in fgf-signaling leads to reduced wnt-signaling  <cit> .
discussion
development of the lung is an intricate process whereby coordination of branching of the epithelium and concomitant differentiation leads to a highly patterned organ.
embryonic lung formation by branching morphogenesis depends on chemotactic attraction by fgf <dig> secreted from the mesenchyme  <cit> .
this is however not the only role of fgf <dig> during lung development.
we here provide evidence that in addition to controlling branching morphogenesis, fgf <dig> maintains the undifferentiated state of the progenitor like cells of the distal buds, hereby coordinating the two processes.
we show here that pdx <dig> is expressed by the epithelium of embryonic lung at e <dig> , and we employ a pdx <dig> promoter construct to drive fgf <dig> expression from this time onwards, as ectopic fgf <dig> expression presumably allows for maintenance of pdx <dig> expression.
notwithstanding a possible physiological role of pdx <dig> in lung development which is not investigated here, the pdx <dig> promoter driven expression of fgf <dig> is sufficient to elicit a significant effect on lung development.
we confirm previous results showing that overexpression of fgf <dig> in the embryonic lung epithelium is highly disruptive for branching morphogenesis  <cit> , and demonstrate that the observed enlargement of the lung is due to hyperplastic growth and formation of large internal cavities.
we conclude that fgf <dig> overexpression has a mitogenic effect in vivo similar to what has been previously demonstrated in vitro  <cit> , corresponding with the reduction of epithelial cell proliferation seen in fgf <dig> hypomorphic mice  <cit> .
transgenic lungs were covered by a dense layer of smooth muscle, also supporting previous findings by mailleux et al  <cit>  that fgf <dig> is required for entry of mesenchymal cells into the parabronchial smooth muscle cell lineage.
based on previous studies of the role of fgf <dig> in more posterior endoderm  <cit>  and the postnatal effect of fgf <dig> overexpression on differentiation of tumors  <cit> , we wished to explore the hypothesis that fgf <dig> functions in embryonic lung progenitor maintenance.
at e <dig> , transgenic pulmonary cells were phenotypically similar to wildtype cells before completion of the canalicular stage .
the majority of the transgenic airway cells remained undifferentiated and mitotic at a time when cells outside of the bronchial epithelium normally cease active proliferation and terminal lung cells differentiate.
markers of early distal cells such as ttf <dig>  the fgfr target pea <dig> and clusterin were co-expressed with pro-surfactant protein c, while markers of differentiated alveolar type i cells and bronchial cells were largely absent.
pro-surfactant protein c is expressed both early in development and in alveolar type ii cells, but mature alveolar type ii cells do not co-express ttf <dig> and clusterin or pea <dig>  in contrast, these markers were co-expressed in the transgenic lung and in wildtype e <dig>  distal bud cells before differentiation.
these results demonstrate that ectopic overexpression of fgf <dig> results in attenuation of differentiation and promotion of a progenitor-like distal cell type.
an embryonic progenitor maintenance program such as this is necessary for sufficient expansion of the progenitor pool before differentiation, but can contribute to oncogenesis if reinstated during postnatal life.
the previous finding that postnatal fgf <dig> overexpression induces lung tumors with high expression of ttf <dig> and sftpc thus corresponds with our findings that fgf <dig> maintains the distal progenitor-like cells during early embryonic development.
consistent with the published fgf <dig> induction of bmp <dig> expression in lung buds in vitro  <cit> , we observed upregulation of bmp <dig> expression in the tg lung.
it is likely that part of the observed effects are caused by the altered expression of bmp <dig>  as bmp signaling has been shown to be necessary for the morphologically correct development of lung buds  <cit> .
however, bmp <dig> has an anti-proliferative effect in the lung, suggesting that the proliferating progenitor-like cells seen in the ppdx1-fgf10flag lung are maintained by a different mechanism.
our results indicate that this mechanism works through activation of beta-catenin.
in previous studies, ectopic expression of an activated form of beta-catenin caused distalization of the proximal airways and goblet cell hyperplasia  <cit> , while ectopic expression of a beta-catenin-lef <dig> fusion protein lead to expansion of progenitor cells and intestinal metaplasia of the lung epithelium  <cit> .
the observed difference between the two studies is probably due to binding specificity of lef <dig> as opposed to tcf factors.
we observe a similar expansion of distal-like progenitor cells, indicating that activation of beta-catenin may be the main mechanism through which fgf <dig> maintains undifferentiated distal cells.
we conclude that the arrested lung phenotype resembles that of the undifferentiated distal lung buds, possibly caused by an increase in bmp <dig> expression and beta-catenin activation, and appears incapable of normal differentiation under conditions of high fgf <dig> expression.
consequently, fgf <dig> elicits lung epithelial progenitor arrest, in a comparable manner to that observed in pancreas  <cit> , stomach  <cit>  and duodenum , indicating a conserved role of fgf <dig> in the endoderm.
in the light of the attenuated lung program, it was possible that transdifferentiation of the lung to a pancreatic, gastric or intestinal fate had occurred as a result of ectopic fgf <dig> and pdx <dig> expression.
respecification of endodermal cells has been noted in several cases.
overexpression of pdx <dig> in the liver of xenopus tadpoles leads to transdifferentiation into pancreas  <cit> , and forced expression of ptf1a and pdx <dig> is capable of inducing a pancreatic fate in more anterior endoderm, including stomach, but not lung  <cit> .
we found no evidence of transdifferentiation using pancreatic markers whereas early lung markers such as ttf <dig> and pro-surfactant protein c were preserved.
there was also no evidence of conversion to a gastric fate.
transdifferentiation of lung epithelium to an intestine-like fate as seen in the beta-catenin-lef <dig> fusion protein model resulted in expression of paneth cell markers, math <dig> and cdx <dig> among other intestinal genes <cit> .
although fgf <dig> overexpression leads to dephosphorylation and activation of beta-catenin in our model, we did not find evidence to suggest a major transformation towards an intestinal fate.
although we did observe a few math <dig> expressing cells in the lung, and also several goblet cells, which derive from math <dig> expressing cells, the absence of cdx <dig> and fabp <dig> argues against major posteriorization of the lung to an intestinal phenotype.
our observation that goblet cells were present in the prospective alveolar epithelium is indicative of goblet cell metaplasia.
increased presence of goblet cells in the lung is often observed during adult lung pathogenesis such as asthma, copd and cystic fibrosis.
adult goblet cell hyperplasia has been associated with activation of egfr resulting in signaling through the ras-map-kinase pathway leading to an increase in expression of genes involved in goblet cell formation .
it is possible that increased fgfr signaling upstream of the map-kinase pathway may trigger the same mechanistic pathway, consistent with our observation of upregulation of pea <dig>  a target of the map-kinase pathway.
an alternate mechanism could be that the fgf <dig> mediated activation of beta-catenin seen in our model lead to goblet cell metaplasia, as expression of an activated form of beta-catenin results in goblet cell hyperplasia of the bronchial epithelium through this mechanism  <cit>  goblet cell metaplasia has been shown to originate from clara cells  in adult lung pathogenesis.
it seems highly unlikely that goblet cells originate from clara cells in our model, as we did not observe any goblet cells near the bronchial epithelium.
alveolar type ii cells function as a progenitor cell for the alveolar cells  <cit> , raising the possibility that metaplastic goblet cells could have differentiated from alveolar type ii progenitor cells.
we cannot exclude this possibility, although no sftpc and dba double-positive cells were found.
we conclude that fgf <dig> overexpression leads to embryonic goblet cell metaplasia of the prospective alveolar epithelium, but not conversion to a more posterior endodermal fate.
these results demonstrate that, even though fgf <dig> overexpression perturbs lung morphology and attenuates the lung differentiation program, the lung still retains pulmonary identity.
CONCLUSIONS
fgf <dig> is normally expressed in proximity to budding epithelium, where it directs branching morphogenesis spatially  <cit> .
after a certain degree of completion of the branching process, differentiation occurs into either proximal or distal lung regions, as a response to intra-organ patterning set up early in lung development.
our results here demonstrate that overexpression of fgf <dig> during embryonic development leads to attenuation of differentiation, distalization of the epithelium and goblet cell hyperplasia.
these results suggest that a single molecule, fgf <dig>  is intricately involved in coordinating pulmonary branching morphogenesis and cellular differentiation.
in turn, this argues for a possible coordinate regulation of these two processes, which perhaps therefore should not be viewed as separate mechanisms.

 the lung forms as two evaginations from the ventral foregut at e, a few days after the initial anterior to posterior specification of the uniform gut tube takes place. sequential branching of the epithelium forms an intricate tree of airways with a distinct axis of proximal to distal differentiation, and a coordinated formation of blood vessels at the distal end. an understanding of the budding process that leads to epithelial branching is quite advanced. organ culture experiments have shown that branching morphogenesis depends on the presence of lung mesenchyme, which induces branching in tracheal epithelium, and that the mesenchymal-expressed fibroblast growth factor can substitute for mesenchyme. the importance of fgf for lung development is demonstrated by the fact that fgf null mice die at birth due to numerous defects, one of them being the absence of lung buds. using lung explant culture bellusci et al and park et al demonstrated that fgf acts as a chemoattractant for the epithelium in lung buds in vitro. fgf expression studies suggest that fgf10-signaling plays an iterative role during lung branching morphogenesis in vivo, as fgf is expressed in a dynamic pattern at the tip of each forming bud. although it is still unknown precisely how this fgf expression pattern is controlled, factors that regulate fgf expression in the lung include fgf, tgf-beta, shh and bmp, and interplay between the budding epithelium and the mesenchyme causes a rapid downregulation of fgf as soon as budding is initiated. in vivo studies of the mechanism by which fgf regulates development of the lung are limited, but a study employing transgenic overexpression of fgf by promoter elements from clara cell secretory protein or surfactant protein c genes demonstrated a pronounced prenatal and postnatal effect of fgf overexpression. transgenic expression could be detected starting from e after embryonic induction and resulted in perturbed branching morphogenesis and formation of adenomatous malformations in e lungs, demonstrating that localized dynamic expression is the key to proper branching. postnatal overexpression of fgf led to tumor formation and differentiation of epithelial tumor cells into a distal type ii alveolar cell like phenotype. affected areas regressed into proximal cell types upon fgf cessation indicating that the differentiated distal cells in this transgenic setting required ongoing fgf signaling to be maintained. this result indicates that fgf may play a role in selectively regulating differentiation of the two different pulmonary cell lineages; the proximal conducting airways and the distal respiratory airways. the bronchial epithelium contains clara cells, ciliated cells and neuroendocrine cells, while the alveolar epithelium consists of type i and type ii alveolar cells. since most of these cell types are specified already before birth, and ectopic fgf has the potential to balance cell types in favor of distal cells postnatally, it is possible that fgf plays a role in this prenatal specification, although this question has not yet been addressed. fgf has been shown to regulate prenatal differentiation in other endodermal organs and act as a progenitor maintenance factor. in the pancreas, fgf null mice are depleted of progenitor cells leading to arrest of growth and differentiation of the epithelium, while overexpression causes expansion of progenitor cells and attenuation of differentiation. a similar function is seen in the gastrointestinal tract, as fgf null mice exhibit a decrease in dividing progenitor cells, and ectopic overexpression leads to attenuation of differentiation in a lineage specific manner. the apparent dichotomy of an established role of fgf as a chemoattractant in the lung versus a progenitor maintenance factor in the gastrointestinal organs might be rooted in lack of available information due to the dramatic early phenotype in the lungs of fgf null mice. indeed, a recent paper has demonstrated that dosage of fgf in the lungs is critical for progenitor cell amplification. hypomorphic lungs exhibit severe hypoplasia most likely due to insufficient progenitor cell maintenance and reduced vascular development. during the course of our analyses of the role of fgf on more posterior endoderm, based on a model of ectopic expression of fgf using the supposedly pancreas/duodenal/gastric specific pdx promoter element, we surprisingly detected a dramatic effect on lung development. the transgenic model allowed us to address the hypothesis that fgf controls differentiation in the embryonic lung epithelium and functions in lung progenitor maintenance. we here demonstrate that the endogenous pdx protein is expressed in developing lung at reduced levels compared to the previously described expression in posterior stomach, pancreas and anterior duodenum, and we find elevated level of fgf mrna and protein in lungs of the ppdx1-fgf model from e. we observe effects on multiple aspects of lung development upon ectopic presence of fgf within the developing lung epithelium: our studies confirm that epithelial branching is impaired in fgf overexpressing lung lobes. additional effects in the embryonic lung include increased cell proliferation, distalization of the epithelium, attenuation of terminal differentiation, and goblet cell metaplasia. our work using a gain-of-function approach suggests that the tight control of fgf expression observed in the lung is important in controlling the balance between proliferation and differentiation as well as cell lineage determination.  fgf10flag is expressed in lung lobes of ppdx1-fgf10flag mice transient transgenic mice expressing a flag-tagged version of mouse fgf under the pdx promoter elements were obtained by oocyte injection as previously described. although this pdx promoter is thought to be specific to the pancreas, duodenum and posterior stomach, we found a profound phenotype in the lung in approximately half of the transient transgenic mice, causing perinatal lethality. initial assessment of ppdx1-fgf10flag embryos with malformed lungs revealed enlargement of one or more lung lobes, and an increase in the distance between terminal branches. the phenotype varied between individual mice; in two of the transgenic mice analyzed by histochemistry the entire lung was aberrantly formed, while the other two embryos had abnormal formation of only one of the right lobes or the entire left lobe. immunohistochemistry for fgf using an antibody that only detects high amounts of protein, as well as an antibody for the flag-tag demonstrated that epithelial cells in these lung lobes contained transgenic fgf while the normal lobes did not. some cells displayed abundant fgf expression in the entire cytoplasm, suggesting that these cells synthesize fgf while the majority of cells only stained for fgf in the cell membrane. in order to validate the production of transgenic fgf in the lung itself, we performed in situ hybridization for fgf and low cycle multiplex rt-pcr for fgf10flag mrna using g6pd- as a control. mpx rt-pcr for the transgenic construct showed that fgf10flag mrna was detectable in the tg lung. in situ hybridization with a short detection time showed fgf in the epithelium of the transgenic lung lobes of e and e embryos, confirming ectopic presence of fgf the highest fgf mrna expression was confined to small clusters of cells in the e embryos and to isolated cells at e. variation in expression of fgf in pancreas and stomach due to mosaicism in these embryos has been reported before, in all cases, fgf protein expression was widespread due to the secreted nature of the protein. the observed phenotype and the expression of fgf10flag mrna and protein in the transgenic lung demonstrate that the pdx promoter construct must be active in this tissue. this was surprising given the previously reported very accurate replication of endogenous pdx expression conferred by the construct in the pancreas, duodenum and posterior stomach. as the pdx promoter fragment used is limited to app. kb upstream regulatory sequence, lung expression may be explained by absence of regulatory elements involved in repression of the pdx promoter outside the distal foregut domain. however, it was also possible that pdx is normally expressed in lung at a low level and would therefore not have been previously detected. we tested this latter hypothesis by performing an analysis of pdx expression throughout embryonic development. our results revealed that pdx protein is present in the early lung buds at e, albeit at a very low level compared to pancreatic and duodenal expression. we were unable to detect any pdx expression in e and e wildtype lungs, but in transgene expressing lung lobes significant pdx protein expression was maintained in clusters of epithelial cells. these lobes exhibited the transgenic phenotype in the entire lobe, likely attributed to the secreted nature of fgf not all pdx expressing cells displayed high levels of cytoplasmic staining for fgf but all fgf secreting cells were positive for pdx. these observations argue that fgf is capable of autocrine maintenance of cells with pdx expression, otherwise lost during development. disruption of branching morphogenesis and bronchiole formation histological examination of transgenic e lungs revealed that the increased size observed was due to hyperplastic growth as well as formation of large empty lumens surrounded by a mesenchymal layer. branching morphogenesis was disrupted leading to formation of dense areas of tissue composed of mesenchyme and epithelium, somewhat resembling the pseudoglandular stage. proximal bronchi were formed to some degree, but terminal bronchioles were absent. these results support previous findings that fgf overexpression in the lung is lethal at birth due to pulmonary defects including perturbed branching morphogenesis. in embryos with only one malformed lobe, the surrounding lobes appeared normal. throughout this study we show data obtained from one embryo with a single malformed lobe as well as data obtained from one transgenic embryo with the entire lung malformed and one wildtype littermate embryo, as each was representative for their phenotype. as we cannot exclude that wildtype-like lobes in the transgenic embryos have been exposed to ectopic fgf early in development, the tg lung lobes displaying wildtype morphology are labeled "wt" on the figures also containing tg lung lobes with a pronounced phenotype. fgf overexpression results in lung hyperplasia due to increased cell proliferation the number of cells undergoing mitosis at e in the alveolar tissue is normally negligible, although the entire epithelium in the bronchi is still positive for proliferating cell nuclear antigen. in contrast, the majority of cells were pcna positive throughout the transgenic lung lobes, in mesenchyme and epithelium, demonstrating that transgenic lung lobes are still in a proliferative state. indeed, as judged from phosphorylated histone h staining, many cells were in m-phase in the tg lobe, but very few in the "wt" lobe. these results support previous findings that fgf stimulates pulmonary proliferation in vitro and correspond with the reduction of epithelial cell proliferation seen in fgf hypomorphic mice. fgf promotes formation of smooth muscle fgf is expressed in parabronchial smooth muscle progenitors and mice hypomorphic for fgf and mice with a fgfr2b splicing defect exhibit a reduction in smooth muscle cells. we therefore proceeded to analyze expression of smooth muscle actin by immunohistochemistry. parabronchial smooth muscle cells seemed unaltered in those cases where proximal bronchi did form, but unlike wildtype lungs, transgenic lungs had developed a layer of smooth muscle actin positive cells surrounding the perimeter of the lung lobe. a few cells within this band were actively dividing. attenuation of alveolar differentiation and retention of markers of the undifferentiated distal buds the high number of cells still undergoing mitosis at e indicated that transgenic pulmonary cells had not differentiated to the same extent as wildtype. pro-surfactant protein c is expressed as early as e in the murine lung progenitor cells and is also expressed by alveolar type ii cells developing before birth. this protein was expressed in both tg and wt lungs, but was expressed in cuboidal cells arranged in a simple epithelium in the transgenic lobes as opposed to the rounded solitary alveolar type ii cells in wt lungs. the majority of transgenic epithelial cells were pro-surfactant protein c positive. analysis of expression of the receptor for advanced glycation end-products, a marker for alveolar type i cells revealed an almost complete absence in tg lungs. type i alveolar cells are abundant in e wildtype lungs and constitute a major part of the alveolar epithelium, with type ii alveolar cells occupying the corners between type i cells. the absence of type i cells in the transgenic lobes and the presence of pro-surfactant protein c expressing cells that did not resemble mature type ii cells, suggested that differentiation was inhibited. further characterization of the pro-surfactant protein c expressing cells showed that these cells were actively dividing, and that they expressed the ets factor pea, which is a fgfr-signaling target and a marker of distal bud cells. this transcription factor was only expressed in a few cells in the wildtype alveolar tissue, and was not expressed in type ii cells. in order to determine if fgf overexpression had resulted in arrest of pulmonary cells in a state resembling the undifferentiated distal bud cells, as suggested by pea expression, we analyzed the presence of several other markers that characterize these cells. ttf encodes a lung-specific homeodomain factor that is expressed from e in the lung bud epithelium and after birth in type ii cells. we detected presence of ttf in transgenic epithelial cells, but only in a few peripheral wildtype cells. clusterin is a glycoprotein that is associated with cell damage in adult cells and with differentiation during development. it is expressed transiently during branching morphogenesis in the lung and is localized in the epithelial cells of the distal buds. clusterin was not expressed in wt e lung, but was expressed in transgenic epithelial cells facing the lumen. ttf clusterin and pro-surfactant protein c were co-expressed by the transgenic epithelial cells, similar to what is seen in the distal tips during the late pseudoglandular stage. the observed expansion of the expression pattern of several early distal markers indicate that fgf overexpression leads to distalization of the entire embryonic lung, similar to what has previously been found in postnatal lung tumors. the absence of mature distal type i and type ii cells furthermore indicate that the transgenic cells, although specified to a distal fate, are arrested in a progenitor-like state. bronchi and bronchiole formation and differentiation are disrupted by fgf10 initial assessment of the phenotype revealed that terminal bronchioles were missing, while proximal bronchi development had taken place to some degree, but had differentiation of the bronchial cell lineages taken place? analysis for expression of uteroglobin, a clara cell marker, revealed that, as expected from the lack of terminal bronchiole development, there were no clara cells in the distal lung. proximal bronchi development was also perturbed as the number of cells expressing uteroglobin was reduced compared to wildtype. sox is expressed in ciliated cells of wildtype trachea and bronchi, and expression of this marker was greatly reduced by fgf ciliated cells were found in the most proximal areas, but were similarly reduced as the clara cells, while they were almost absent from more distal areas. only a few scattered ciliated cells remained in the distal epithelium suggesting that bronchiole development was aborted. interestingly, one particular bronchial lineage descendant was capable of forming. cgrp expressing pulmonary neuroendocrine cells were still present in the transgenic lung lobes. in the wildtype lung these cells were confined to bronchi and bronchioles and appeared in small clusters, while they were scattered throughout the transgenic lung. our results support the assessment that bronchiole development was terminated, as only pnecs were present in the distal transgenic lung. expression of pdx in the lung does not lead to transdifferentiation to intestinal, gastric or pancreatic fates the observation that transgenic lungs lacked terminal pulmonary cells lead to speculations of whether transdifferentiation to a pancreatic, gastric or intestinal fate had taken place, due to ectopic expression of fgf and pdx we performed immunohistochemistry for expression of nkx and insulin, but did not detect expression, indicating that transdifferentiation to a pancreatic fate did not occur. cdx is expressed throughout the intestine, math is expressed by intestinal secretory cell lineages, cryptdin by paneth cells and intestinal fatty acid binding protein by enterocytes. in the transgenic lungs we could not detect cdx or fabp expression, and only very few cryptdin or math expressing cells. sox is expressed in the gastric epithelium, throughout the esophagus, and is present in the ciliated cells in the bronchi, but very little expression was seen in the transgenic tissue outside of bronchi. likewise, h/k-atpase and pepsinogenc, expressed by parietal and chief cells respectively, were also absent. trefoil factor is normally expressed by mucous producing cells in the stomach, and is absent from embryonic lung. a few tff positive cells were found in one out of three transgenic mice. tff is expressed in intestinal goblet cells, and although not expressed in embryonic lung, is expressed by pulmonary goblet cells in the tracheal submucosal glands in adult mice. tff was expressed in several cells throughout the distal epithelium in all transgenic lobes. we therefore conclude that transdifferentiation to a gut like fate had not taken place, although limited expression of math and tff as well as high expression of tff pointed towards a possible goblet cell metaplasia. embryonic goblet cell metaplasia goblet cells are present in only few numbers in submucosal glands of adult mice although more cells can form as a response to inflammation in adult mice and humans, and in some human lung cancers. goblet cells react with dolichos biflorus agglutinin, and detection of rhodamine or cy conjugated dba, as well as pas/alcian blue histochemistry, confirmed that goblet cells were present in the distal epithelium of transgenic but not wildtype lobes. no dba reactivity was found in bronchi or trachea. since this goblet cell metaplasia had arisen in the prospective alveolar epithelium, we performed a double immunofluorescence analysis for dba and pro-surfactant protein c. similar to wt lung, dba and p-sftpc were not co-expressed. increased beta-catenin signaling and bmp expression regulation of transcription through activation of stabilized beta-catenin is necessary for differentiation of distal airways, and transgenic expression of an activated form of beta-catenin leads to goblet cell metaplasia and expansion of alveolar type ii-like cells. since goblet cell metaplasia and expansion of surfactant protein c expressing cells were also observed in the ppdx1-fgf10flag model, we next tested whether the observed phenotype could be caused partly through activation of beta-catenin. we analyzed for activation of beta-catenin using an antibody specific for the dephosphorylated serine or threonine which is an activated form. cells positive for activated beta-catenin were present throughout the epithelium and mesenchyme in both wildtype and transgenic lobes, and more activated cells were present in the distal part of the wildtype lung, than the proximal part. the frequency of activated beta-catenin cells was higher in the transgenic lobes, especially in the proximal lung but also in the distal lung, suggesting that fgf overexpression leads to activation of beta-catenin. bmp is expressed in the distal-most epithelial cells in proximity to the fgf expressing mesenchyme, and bmp expression is upregulated in response to a fgf bead. we therefore analyzed whether bmp expression was increased in the ppdx1-fgf10flag lung. in situ hybridization revealed that bmp was expressed in scattered epithelial cells at e, while the number of bmp positive cells was increased in transgenic lungs. this result was confirmed by mpx rt-pcr, where bmp but not bmp expression was highly upregulated in transgenic lungs. these results confirm previous in vitro data and demonstrate that fgf overexpression leads to activation of beta-catenin and upregulation of epithelial bmp expression, suggesting that these are downstream effectors of fgf and could be involved in the observed phenotype. this observation is in agreement with previous reports showing that reductions in fgf-signaling leads to reduced wnt-signaling. discussion development of the lung is an intricate process whereby coordination of branching of the epithelium and concomitant differentiation leads to a highly patterned organ. embryonic lung formation by branching morphogenesis depends on chemotactic attraction by fgf secreted from the mesenchyme. this is however not the only role of fgf during lung development. we here provide evidence that in addition to controlling branching morphogenesis, fgf maintains the undifferentiated state of the progenitor like cells of the distal buds, hereby coordinating the two processes. we show here that pdx is expressed by the epithelium of embryonic lung at e, and we employ a pdx promoter construct to drive fgf expression from this time onwards, as ectopic fgf expression presumably allows for maintenance of pdx expression. notwithstanding a possible physiological role of pdx in lung development which is not investigated here, the pdx promoter driven expression of fgf is sufficient to elicit a significant effect on lung development. we confirm previous results showing that overexpression of fgf in the embryonic lung epithelium is highly disruptive for branching morphogenesis, and demonstrate that the observed enlargement of the lung is due to hyperplastic growth and formation of large internal cavities. we conclude that fgf overexpression has a mitogenic effect in vivo similar to what has been previously demonstrated in vitro, corresponding with the reduction of epithelial cell proliferation seen in fgf hypomorphic mice. transgenic lungs were covered by a dense layer of smooth muscle, also supporting previous findings by mailleux et al that fgf is required for entry of mesenchymal cells into the parabronchial smooth muscle cell lineage. based on previous studies of the role of fgf in more posterior endoderm and the postnatal effect of fgf overexpression on differentiation of tumors, we wished to explore the hypothesis that fgf functions in embryonic lung progenitor maintenance. at e, transgenic pulmonary cells were phenotypically similar to wildtype cells before completion of the canalicular stage. the majority of the transgenic airway cells remained undifferentiated and mitotic at a time when cells outside of the bronchial epithelium normally cease active proliferation and terminal lung cells differentiate. markers of early distal cells such as ttf the fgfr target pea and clusterin were co-expressed with pro-surfactant protein c, while markers of differentiated alveolar type i cells and bronchial cells were largely absent. pro-surfactant protein c is expressed both early in development and in alveolar type ii cells, but mature alveolar type ii cells do not co-express ttf and clusterin or pea in contrast, these markers were co-expressed in the transgenic lung and in wildtype e distal bud cells before differentiation. these results demonstrate that ectopic overexpression of fgf results in attenuation of differentiation and promotion of a progenitor-like distal cell type. an embryonic progenitor maintenance program such as this is necessary for sufficient expansion of the progenitor pool before differentiation, but can contribute to oncogenesis if reinstated during postnatal life. the previous finding that postnatal fgf overexpression induces lung tumors with high expression of ttf and sftpc thus corresponds with our findings that fgf maintains the distal progenitor-like cells during early embryonic development. consistent with the published fgf induction of bmp expression in lung buds in vitro, we observed upregulation of bmp expression in the tg lung. it is likely that part of the observed effects are caused by the altered expression of bmp as bmp signaling has been shown to be necessary for the morphologically correct development of lung buds. however, bmp has an anti-proliferative effect in the lung, suggesting that the proliferating progenitor-like cells seen in the ppdx1-fgf10flag lung are maintained by a different mechanism. our results indicate that this mechanism works through activation of beta-catenin. in previous studies, ectopic expression of an activated form of beta-catenin caused distalization of the proximal airways and goblet cell hyperplasia, while ectopic expression of a beta-catenin-lef fusion protein lead to expansion of progenitor cells and intestinal metaplasia of the lung epithelium. the observed difference between the two studies is probably due to binding specificity of lef as opposed to tcf factors. we observe a similar expansion of distal-like progenitor cells, indicating that activation of beta-catenin may be the main mechanism through which fgf maintains undifferentiated distal cells. we conclude that the arrested lung phenotype resembles that of the undifferentiated distal lung buds, possibly caused by an increase in bmp expression and beta-catenin activation, and appears incapable of normal differentiation under conditions of high fgf expression. consequently, fgf elicits lung epithelial progenitor arrest, in a comparable manner to that observed in pancreas, stomach and duodenum, indicating a conserved role of fgf in the endoderm. in the light of the attenuated lung program, it was possible that transdifferentiation of the lung to a pancreatic, gastric or intestinal fate had occurred as a result of ectopic fgf and pdx expression. respecification of endodermal cells has been noted in several cases. overexpression of pdx in the liver of xenopus tadpoles leads to transdifferentiation into pancreas, and forced expression of ptf1a and pdx is capable of inducing a pancreatic fate in more anterior endoderm, including stomach, but not lung. we found no evidence of transdifferentiation using pancreatic markers whereas early lung markers such as ttf and pro-surfactant protein c were preserved. there was also no evidence of conversion to a gastric fate. transdifferentiation of lung epithelium to an intestine-like fate as seen in the beta-catenin-lef fusion protein model resulted in expression of paneth cell markers, math and cdx among other intestinal genes. although fgf overexpression leads to dephosphorylation and activation of beta-catenin in our model, we did not find evidence to suggest a major transformation towards an intestinal fate. although we did observe a few math expressing cells in the lung, and also several goblet cells, which derive from math expressing cells, the absence of cdx and fabp argues against major posteriorization of the lung to an intestinal phenotype. our observation that goblet cells were present in the prospective alveolar epithelium is indicative of goblet cell metaplasia. increased presence of goblet cells in the lung is often observed during adult lung pathogenesis such as asthma, copd and cystic fibrosis. adult goblet cell hyperplasia has been associated with activation of egfr resulting in signaling through the ras-map-kinase pathway leading to an increase in expression of genes involved in goblet cell formation. it is possible that increased fgfr signaling upstream of the map-kinase pathway may trigger the same mechanistic pathway, consistent with our observation of upregulation of pea a target of the map-kinase pathway. an alternate mechanism could be that the fgf mediated activation of beta-catenin seen in our model lead to goblet cell metaplasia, as expression of an activated form of beta-catenin results in goblet cell hyperplasia of the bronchial epithelium through this mechanism goblet cell metaplasia has been shown to originate from clara cells in adult lung pathogenesis. it seems highly unlikely that goblet cells originate from clara cells in our model, as we did not observe any goblet cells near the bronchial epithelium. alveolar type ii cells function as a progenitor cell for the alveolar cells, raising the possibility that metaplastic goblet cells could have differentiated from alveolar type ii progenitor cells. we cannot exclude this possibility, although no sftpc and dba double-positive cells were found. we conclude that fgf overexpression leads to embryonic goblet cell metaplasia of the prospective alveolar epithelium, but not conversion to a more posterior endodermal fate. these results demonstrate that, even though fgf overexpression perturbs lung morphology and attenuates the lung differentiation program, the lung still retains pulmonary identity.  fgf is normally expressed in proximity to budding epithelium, where it directs branching morphogenesis spatially. after a certain degree of completion of the branching process, differentiation occurs into either proximal or distal lung regions, as a response to intra-organ patterning set up early in lung development. our results here demonstrate that overexpression of fgf during embryonic development leads to attenuation of differentiation, distalization of the epithelium and goblet cell hyperplasia. these results suggest that a single molecule, fgf is intricately involved in coordinating pulmonary branching morphogenesis and cellular differentiation. in turn, this argues for a possible coordinate regulation of these two processes, which perhaps therefore should not be viewed as separate mechanisms. 
through gain-of-function analysis, we here find that fgf inhibits differentiation of the lung epithelium and promotes distalization of the embryonic lung. we conclude that fgf inhibits terminal differentiation in the embryonic lung and maintains the distal epithelium, and that excessive levels of fgf leads to metaplastic differentiation of goblet cells similar to that seen in chronic inflammatory diseases. the transgenic epithelium consisted predominantly of proliferating progenitor-like cells expressing pro-surfactant protein c, ttf pea and clusterin similarly to immature distal tip cells. previous studies have shown that this requirement for fgf is due in part to its role as a chemotactic factor during branching morphogenesis. ectopic expression of fgf in the lung epithelium caused impaired lung development and perinatal lethality in a transgenic mouse model. interaction with the surrounding mesenchyme is necessary for development of endodermal organs, and fibroblast growth factors have recently emerged as mesenchymal-expressed morphogens that direct endodermal morphogenesis. in other endodermal organs fgf also plays a role in regulating differentiation. lung lobes were enlarged due to increased interlobular distance and hyperplasia of the airway epithelium. strikingly, goblet cells developed within this arrested epithelium leading to goblet cell hyperplasia.
 interaction with the surrounding mesenchyme is necessary for development of endodermal organs, and fibroblast growth factors have recently emerged as mesenchymal-expressed morphogens that direct endodermal morphogenesis. the fibroblast growth factor null mouse is characterized by the absence of lung bud development. previous studies have shown that this requirement for fgf is due in part to its role as a chemotactic factor during branching morphogenesis. in other endodermal organs fgf also plays a role in regulating differentiation.  through gain-of-function analysis, we here find that fgf inhibits differentiation of the lung epithelium and promotes distalization of the embryonic lung. ectopic expression of fgf in the lung epithelium caused impaired lung development and perinatal lethality in a transgenic mouse model. lung lobes were enlarged due to increased interlobular distance and hyperplasia of the airway epithelium. differentiation of bronchial and alveolar cell lineages was inhibited. the transgenic epithelium consisted predominantly of proliferating progenitor-like cells expressing pro-surfactant protein c, ttf pea and clusterin similarly to immature distal tip cells. strikingly, goblet cells developed within this arrested epithelium leading to goblet cell hyperplasia.  we conclude that fgf inhibits terminal differentiation in the embryonic lung and maintains the distal epithelium, and that excessive levels of fgf leads to metaplastic differentiation of goblet cells similar to that seen in chronic inflammatory diseases. 