BACKGROUND
the preparation of the lung for an aerobic environment includes the surge of surfactant synthesis, which occurs late in pregnancy in type ii pneumocytes  in the distal epithelium .
pulmonary surfactant is a combination of lipids and proteins  <cit>  enabling normal respiration by preventing alveolar collapse.
surfactant deficiency is the major cause of respiratory distress syndrome of the neonate   <cit> , a pathology occurring when birth arises before adequate ptii cell maturation.
fatty acids are precursor molecules in the synthesis of surfactant phospholipids.
they can be synthesized in the lung or originate from circulating triglycerides.
in the plasma, triglycerides are mainly found in the core of vldl and chylomicrons, the latter carrying alimentary lipids after secretion by the small intestine.
in many tissues including adipose tissue and skeletal muscle, delivery of fatty acids from triglyceride-rich lipoproteins occurs by hydrolysis on the luminal surface of the capillary endothelium.
this reaction is catalyzed by lipoprotein lipase   <cit>  and requires apolipoprotein c-ii  as essential and specific cofactor  <cit> .
lpl expression was studied in the mature lung.
in the guinea pig, lpl mrna was mainly found in alveolar macrophages, while the protein was mainly localized in capillaries  <cit> .
this is compatible with a previous observation in the adult rat where lpl activity was found in lung macrophages  <cit> .
in the human, fetal lung explants from the second trimester of gestation were studied  <cit> .
lpl protein was found at the surface of epithelial cells after stimulation of the tissue with dexamethasone/8-br-camp/isobutylmethylxanthine.
recently, we reported expression of lpl and apoc-ii in the fetal mouse lung between gestation days   <dig>  to  <dig>   <cit> .
a sex difference in the level of apoc-ii mrna was observed , while a significant increase in lpl mrna was found from gd  <dig>  to  <dig>  .
immunohistochemistry  revealed the presence of apoc-ii in secretory granule-like structures in the distal epithelium, mainly near the basal membrane, close to the mesenchyme, a structure that is distinct from lamellar bodies  <cit> .
the fact that the apoc-ii protein is found at this site on the day when the surge of surfactant synthesis occurs suggests the participation of apoc-ii in this process.
many questions arise from this work for which an answer will clarify the role of apoc-ii and lpl in surfactant synthesis.
does apoc-ii protein localization change according to the stage of lung development, thus according to the need in surfactant?
are lpl molecules translocated to the luminal surface of capillaries?
do the sites of apoc-ii and lpl gene expression change according to the stage of lung development and to protein localization?
to answer these questions, we have performed in situ hybridization  and ihc of lpl and apoc-ii from gd  <dig>  to the first days of alveolarization.
qpcr analysis of apoc-ii and lpl mrnas was also performed with samples from gd  <dig>  to postnatal day  <dig> .
RESULTS
mrna and protein localization of lpl and apoc-ii during the pseudoglandular and the canalicular stages
the surge of surfactant synthesis occurs on gd  <dig>  in the mouse as indicated by the appearance of lamellar bodies  <cit> , an increase in surface activity in the mouse lung homogenate  <cit> , and by increases in the activity of some enzymes involved in pulmonary lipid metabolism  <cit> .
therefore, we have first studied samples from pseudoglandular  to late canalicular  stages of lung development in order to determine whether mrna and/or protein localization of lpl and apoc-ii change in preparation to the surge of surfactant synthesis.
on gd  <dig> , apoc-ii mrna was found mainly in the mesenchyme .
positive signals were also found in cells of some epithelia , while the most proximal epithelium was negative .
in contrast, the apoc-ii protein did not accumulate in all the sites of apoc-ii gene expression on gd  <dig>  but only in the epithelium where a diffuse signal was observed in the cytoplasm .
on gd  <dig> , the apoc-ii mrna and protein co-localized.
they were found only in the distal epithelium with lumina  and the most distal epithelium before apparition of the lumina .
it should be noted that structures corresponding to the most distal epithelium differ between gd  <dig>  and  <dig> .
the most distal epithelium observed on gd  <dig>  is no longer the most distal epithelium on gd  <dig>  where a more differentiated epithelium is observed.
the identity of cells composing the most distal epithelium with no lumina on gd  <dig>  was confirmed by ihc using an anti-cytokeratin  <dig> antibody , which is a marker of epithelial cells  <cit> .
recently we reported that the apoc-ii protein accumulated on gd  <dig>  in the distal epithelium, more precisely in structures looking like secretory granules, mainly localized near the basal membrane, close to the mesenchyme  <cit> .
similar results were obtained here .
interestingly, we show here that localization of the positive signal in the cells is different on gd  <dig>  compared to gd  <dig> .
no positive secretory granules were observed on gd  <dig> .
accordingly, data obtained on gd  <dig>  show a transition state between results at gd  <dig>  and those at gd  <dig> .
as shown in figure 1i, both a diffuse positive signal in the cytoplasm of epithelial cells and a few dots looking like secretory granules were observed in the same tissue.
figure 1j is from another gd  <dig>  litter and shows small positive dots, but no diffuse signal in the epithelium, which could represent a later developmental event.
no positive signal was observed by ish using lung materials of  <dig> fetuses from  <dig> litters sacrificed on gd  <dig>  , which is also compatible with a transition state.
lpl protein was found in capillary-like structures on gd  <dig> , gd  <dig> , and gd  <dig>  .
lpl mrna was mainly found in epithelial cells of the distal epithelium on gd  <dig>  .
this mrna was absent of the proximal epithelium on gd  <dig>  except for a few cells that may present barely detectable signals .
on gd  <dig> , lpl mrna was mainly found in the mesenchyme and the distal epithelium  as for apoc-ii mrna.
on gd  <dig> , no signal was obtained by ish for lpl using lung tissues of  <dig> fetuses from  <dig> litters .
in order to confirm that the lpl-positive signal corresponds to capillaries and not to α-smooth muscle actin -positive structures, an anti-platelet endothelial cell adhesion molecule- <dig>  antibody was used on gd  <dig> -lung tissue sections to stain capillaries.
results show that staining of capillary network  is very similar to the positive signals identified as capillary-like structures using an anti-lpl antibody.
in contrast, the use of an anti-α-sma antibody leads to a different staining profile .
apoc-ii  and lpl  mrnas were observed in smooth muscles surrounding large arteries.
lpl protein was found in smooth muscles of arteries , but signal intensities were lower than those found in adjacent capillaries.
no ihc signal was found in smooth muscles for apoc-ii .
in most cases, it was difficult to determine whether endothelial cells of large vessels were stained because of the proximity to positive structures.
nevertheless, endothelial cells positive for lpl by ihc  and others negative for apoc-ii by ish and ihc  were also found on gd  <dig> .
apoc-ii and lpl mrna levels during the saccular stage and the first segment of the alveolar stage
apoc-ii and lpl qpcr data were recently published for samples from gd  <dig>  to gd  <dig>   <cit> , but not for further developmental times.
here, qpcr analysis of these two genes is presented between gd  <dig>  and pn  <dig> using male and female pools of various litters .
levels of apoc-ii mrna showed more variations from sample to sample than those of lpl mrna.
there was no statistically significant sex difference for the two genes, even though higher values were observed for males for apoc-ii mrna on pn  <dig> in four of the five analyzed litters .
a statistically significant difference according to developmental time was observed only for lpl mrna  .
mrna and protein localization of lpl and apoc-ii during the saccular and the beginning of the alveolar stages of lung development
similarities were found between apoc-ii and lpl expression patterns during the perinatal period.
both genes were expressed in the distal epithelium short time before birth , but not after birth  .
in contrast, the proximal epithelium was negative on gd  <dig> , showed a weak positive signal on gd  <dig>  and  <dig>  , presented a marked increase in intensity soon after birth , and was negative on pn  <dig> for both genes.
using  <dig> fetuses on gd  <dig>   and  <dig> neonates on pn  <dig>  we confirmed that the switch from the distal to the proximal epithelium correlated with birth, not with sex .
apoc-ii and lpl mrnas were also found in a few scattered distal epithelial cells after birth.
in addition, positive signals by ish were observed in association with blood vessels before birth  but not after birth  for both genes.
on pn  <dig>  thus at junction between saccularization and alveolarization, apoc-ii and lpl mrna were found in alveolar walls , including newly-formed septa .
albeit similarities were observed in the expression profiles of their encoding genes, apoc-ii and lpl proteins did not accumulate in the same structures.
interestingly, apoc-ii was observed in secretory granule-like structures until soon after birth , but positive signal had disappeared one day later .
in fact, between gd  <dig>  and pn  <dig>  apoc-ii-positive secretory granules were mainly observed in distal epithelia with no or small lumina, but not with large lumina .
such a discrepancy was less evident for apoc-ii mrna for which some distal epithelia with large lumina were also positive .
this strongly suggests that apoc-ii protein is synthesized and retained within the epithelium in secretory granules until the lumina reaches a certain diameter, and that it is then secreted .
we did not observe any sex difference or developmental delay in our experiment in that the three males and the three females analyzed on pn  <dig> presented secretory granules, while the six other animals studied on pn  <dig> did not show any secretory granule .
in all of the experiments, neonates sacrificed on pn  <dig> were killed between a few minutes and four hours after the birth of the first neonate.
the apoc-ii protein was still undetectable on pn  <dig> , while secretory granules were found in the epithelial cells of the respiratory bronchioles on pn  <dig>  near the basal membrane, close to the mesenchyme .
only a few secretory granules were still positive on pn  <dig>  apoc-ii-positive capillaries, as those shown on figure 5o, were only observed in one third of the subjects analyzed on pn  <dig>  in fact, after its secretion, apoc-ii protein may be difficult to detect depending of the nature of its association with the tissue.
the situation was clearly different for lpl.
the protein was found in capillaries from pn  <dig> to pn  <dig> for all the analyzed neonates  as well as before birth.
lpl positive signals were also found in cells of the respiratory bronchiole from gd  <dig>  to pn  <dig> , but not on gd  <dig>  and pn  <dig>  in this epithelium, signal intensity showed variation from cell to cell.
in contrast, no protein was detected in most proximal epithelium .
cells localized in the alveolar space on pn  <dig> were positive for apoc-ii and lpl by both ish  and ihc  with no sex difference.
based on their localization and morphology, these cells were most probably macrophages.
apoc-ii and lpl proteins were also found in macrophages on pn  <dig> .
disaturated phosphatidylcholine determination
disaturated phosphatidylcholine  is quantitatively the most important lipid of surfactant  <cit>  and an increase in lung dspc content was observed after the surge of surfactant synthesis  <cit> .
here, relative dspc levels were determined in lungs from balb/c fetal mice from gd  <dig>  to  <dig>  following a one hour mating window .
a marked statistically significant increase was observed between gd  <dig>  and gd  <dig>  .
discussion
surfactant is of the first importance in the distal epithelium to prevent alveolar collapse.
our results show that the lpl-related molecular machinery is regulated temporally and geographically in the developing lung in correlation with the need in surfactant lipid synthesis.
major changes in sites of apoc-ii and lpl mrna are summarized in table  <dig>  changes in protein localization were mainly observed for apoc-ii and are summarized in table  <dig>  our results show that the tissue needing surfactant governs itself lpl and apoc-ii gene expression and apoc-ii secretion during the critical period.
thus, the lpl machinery is subjected to a complex regulation during the canalicular and the saccular stages of lung development.
the need of lipids for surfactant synthesis is particularly important during the period between the surge of surfactant synthesis and birth.
this can explain why the distal epithelium of the lung contributes itself to lpl and apoc-ii synthesis, which must maximizes fatty acid recruitment during this period.
our results are compatible with those of ryan et al  <cit>  showing that maternal loading with vldl stimulates fetal surfactant synthesis.
* apoc-ii was observed in capillaries only in one third of the analyzed subjects on pn 10
one major observation about apoc-ii protein is that it accumulated in secretory granules within several distal epithelia from gd  <dig>  to a developmental time point soon after birth.
thus, a temporal and geographic correlation exists between the appearance of lamellar bodies as reported in the literature  <cit>  and apoc-ii accumulation in secretory granules presented in figure  <dig>  in contrast to apoc-ii, lpl protein was obviously secreted out of the producing cells and accumulated in capillaries.
accordingly, a rapid release of 10-15% of total tissue lpl was observed following infusion of heparin into isolated perfused adult rat lungs .
the presence of lpl in capillaries in the fetal lung is compatible with its recognized function in recruitment of fatty acids from lipoprotein-associated triglycerides  <cit> .
in contrast to lpl, apoc-ii was observed in its producing cells within secretory granules.
this suggests that apoc-ii secretion is controlled, but not at all developmental time points.
for example, no secretory granule is observed on gd  <dig>  indicating that there is no apoc-ii retention within the producing cells.
a transition state is observed on gd  <dig>  with small secretory granules, which must correspond to the retention of newly formed apoc-ii molecules.
from gd  <dig>  to pn  <dig>  distal epithelia with no or small lumina containing apoc-ii-positive secretory granules as well as distal epithelia with large lumina negative for apoc-ii were observed.
as explained in figure  <dig>  these observations suggest that apoc-ii is secreted by distal epithelia reaching a specific level of development, which stimulates local lpl activity for fatty acid recruitment for surfactant synthesis.
in contrast to lpl, apoc-ii was not reported to bind to the endothelium and consequently, most of the apoc-ii molecules secreted by the epithelia would be washed off of the slide during the ihc procedure so that the presence of apoc-ii in capillaries could be difficult to demonstrate.
the fact that apoc-ii is produced by the lung and not only provided by plasma for activation of lpl in capillaries could be explained by the great need for fatty acids for surfactant production and, consequently, the very high amount of lpl molecules necessitating apoc-ii for their activation.
a noticeable increase in surfactant lipid content was observed between gd  <dig>  and  <dig>  .
knowing that surfactant lipids cannot be detected immediately when they start to be produced but have to accumulate over the detection limit before an increase be observed, our results are compatible with the literature showing the appearance of lamellar bodies on gd  <dig>   <cit> .
moreover, the marked increase in dspc levels coincides with the presence of distal epithelium with large lumina without secretory granules, thus after the beginning of apoc-ii secretion for optimization of surfactant lipid production based on our model .
in  <dig>  lpl expression was found in lipofibroblasts isolated from rat developing lungs  <cit> .
more specifically,  <dig> fold and  <dig> fold increases in lpl mrna levels were observed by northern blot analysis in isolated lipofibroblasts and whole lungs, respectively, between postnatal days - <dig> and + <dig>  in our experiments, not only did we fail to reproduce this rise at birth in whole mouse lungs by quantitative real time pcr, but lpl mrna accumulation sites changed from distal to proximal epithelium at birth with no or only a few positive signal in the mesenchyme after birth.
furthermore, the ihc signal we obtained for lpl was close to the background in the mesenchyme during the perinatal period.
of course, it is not excluded that lpl expression in lipofibroblasts be very low and under the limit of detection in our assays, but obviously, our data indicate that this cell population is not a major site of lpl mrna and protein accumulation.
the results of chen et al are also incompatible with other studies reporting many years ago that pulmonary lpl activity in growing rats peaks one day before birth and decreases at birth  <cit> .
the decrease at birth may correspond to the switch we observed in the site of lpl gene expression in the mouse.
in fact, the timing of lpl activity in the rats, the temporal and cell specific regulations of lpl gene expression we observed in the mouse and the presence of lpl in capillaries underscore a role in recruitment of lipids for the surge of surfactant synthesis.
such a role has been already proposed based on the presence of vldl in the fetal circulation at  <dig> days' gestation in the rat when the peak in lpl activity occurs .
lpl proteins were also found in other sites in the developing lung as in respiratory bronchioles during saccularization and in macrophages from the beginning of alveolarization.
as reported for rats, an increase in lpl activity was observed after the first week of life following the decrease at birth  <cit> .
this observation is compatible with the increase we observed in lpl mrna from pn  <dig> to  <dig>  and may be explained by apparition of intra-alveolar macrophages, which express lpl as reported here  and in the literature  <cit> .
our data cannot support or exclude lpl expression by lipofibroblasts at this developmental time.
no sex difference was observed in sites of apoc-ii and lpl gene expression and protein accumulation for any of the analyzed time point.
no sex difference in signal intensity was observed, although ish and ihc are not reliable quantitative tools.
qpcr analysis  did not reveal any significant sex difference when all the population was considered, but some differences from sample to sample prevented us to conclude at the absence of any sex difference at specific time points, as at pn  <dig> when higher levels of apoc-ii mrna were observed more frequently in males.
the sex difference reported recently  <cit>  in apoc-ii mrna levels in favor of females in developmental times preceding gd  <dig>  is not ruled out by the present work.
this statistically significant sex difference in apoc-ii mrna suggests that a significant proportion of females would start accumulating apoc-ii earlier than males.
hypertriglyceridemia is observed in both lpl and apoc-ii deficiencies  <cit> , while there is no evidence that these deficiencies may be associated with a high rate of mortality related to respiratory distress syndrome of the neonate in the human.
the situation is different in lpl knockout mice, which present a high rate of neonatal death caused by respiratory problems  <cit> .
however, the cause of death was engorgement of capillaries by chylomicrons leading to cyanosis, not surfactant insufficiency.
therefore, apoc-ii and lpl seem not essential for obtaining minimal surfactant levels following birth at term.
this situation is not surprising knowing that lpl activity is only one of the two sources of fatty acids, the remaining source being de novo synthesis through fatty acid synthase.
as it is frequently observed for essential mechanisms/metabolisms, a redundancy is observed here in the source of fatty acids for surfactant synthesis.
interestingly, in mice deficient in adipose tissue lpl, fat mass was preserved by large increases in endogenous fatty acid biosynthesis, which compensated for the loss of lpl activity  <cit> .
a similar situation should occur in the developing lung of lpl- and apoc-ii-deficient subjects.
in fact, the importance of the work presented here lies in the fact that preterm birth frequently leads to surfactant insufficiency and therefore, apoc-ii/lpl may become an interesting pharmaceutical target in that context.
CONCLUSIONS
we have shown that sites of apoc-ii as well as lpl mrna and protein accumulation are regulated during the canalicular and the saccular stages of lung development.
these changes show a complex regulation of the lpl molecular machinery during an important period for the adaptation of the lung to gas exchange.
from this work, apoc-ii appears to be an interesting potential pharmaceutical target for treatment and/or prevention of respiratory distress syndrome of the neonate.

 the preparation of the lung for an aerobic environment includes the surge of surfactant synthesis, which occurs late in pregnancy in type ii pneumocytes in the distal epithelium. pulmonary surfactant is a combination of lipids and proteins enabling normal respiration by preventing alveolar collapse. surfactant deficiency is the major cause of respiratory distress syndrome of the neonate, a pathology occurring when birth arises before adequate ptii cell maturation. fatty acids are precursor molecules in the synthesis of surfactant phospholipids. they can be synthesized in the lung or originate from circulating triglycerides. in the plasma, triglycerides are mainly found in the core of vldl and chylomicrons, the latter carrying alimentary lipids after secretion by the small intestine. in many tissues including adipose tissue and skeletal muscle, delivery of fatty acids from triglyceride-rich lipoproteins occurs by hydrolysis on the luminal surface of the capillary endothelium. this reaction is catalyzed by lipoprotein lipase and requires apolipoprotein c-ii as essential and specific cofactor. lpl expression was studied in the mature lung. in the guinea pig, lpl mrna was mainly found in alveolar macrophages, while the protein was mainly localized in capillaries. this is compatible with a previous observation in the adult rat where lpl activity was found in lung macrophages. in the human, fetal lung explants from the second trimester of gestation were studied. lpl protein was found at the surface of epithelial cells after stimulation of the tissue with dexamethasone/8-br-camp/isobutylmethylxanthine. recently, we reported expression of lpl and apoc-ii in the fetal mouse lung between gestation days to. a sex difference in the level of apoc-ii mrna was observed, while a significant increase in lpl mrna was found from gd to. immunohistochemistry revealed the presence of apoc-ii in secretory granule-like structures in the distal epithelium, mainly near the basal membrane, close to the mesenchyme, a structure that is distinct from lamellar bodies. the fact that the apoc-ii protein is found at this site on the day when the surge of surfactant synthesis occurs suggests the participation of apoc-ii in this process. many questions arise from this work for which an answer will clarify the role of apoc-ii and lpl in surfactant synthesis. does apoc-ii protein localization change according to the stage of lung development, thus according to the need in surfactant? are lpl molecules translocated to the luminal surface of capillaries? do the sites of apoc-ii and lpl gene expression change according to the stage of lung development and to protein localization? to answer these questions, we have performed in situ hybridization and ihc of lpl and apoc-ii from gd to the first days of alveolarization. qpcr analysis of apoc-ii and lpl mrnas was also performed with samples from gd to postnatal day.  mrna and protein localization of lpl and apoc-ii during the pseudoglandular and the canalicular stages the surge of surfactant synthesis occurs on gd in the mouse as indicated by the appearance of lamellar bodies, an increase in surface activity in the mouse lung homogenate, and by increases in the activity of some enzymes involved in pulmonary lipid metabolism. therefore, we have first studied samples from pseudoglandular to late canalicular stages of lung development in order to determine whether mrna and/or protein localization of lpl and apoc-ii change in preparation to the surge of surfactant synthesis. on gd, apoc-ii mrna was found mainly in the mesenchyme. positive signals were also found in cells of some epithelia, while the most proximal epithelium was negative. in contrast, the apoc-ii protein did not accumulate in all the sites of apoc-ii gene expression on gd but only in the epithelium where a diffuse signal was observed in the cytoplasm. on gd, the apoc-ii mrna and protein co-localized. they were found only in the distal epithelium with lumina and the most distal epithelium before apparition of the lumina. it should be noted that structures corresponding to the most distal epithelium differ between gd and. the most distal epithelium observed on gd is no longer the most distal epithelium on gd where a more differentiated epithelium is observed. the identity of cells composing the most distal epithelium with no lumina on gd was confirmed by ihc using an anti-cytokeratin antibody, which is a marker of epithelial cells. recently we reported that the apoc-ii protein accumulated on gd in the distal epithelium, more precisely in structures looking like secretory granules, mainly localized near the basal membrane, close to the mesenchyme. similar results were obtained here. interestingly, we show here that localization of the positive signal in the cells is different on gd compared to gd. no positive secretory granules were observed on gd. accordingly, data obtained on gd show a transition state between results at gd and those at gd. as shown in figure 1i, both a diffuse positive signal in the cytoplasm of epithelial cells and a few dots looking like secretory granules were observed in the same tissue. figure 1j is from another gd litter and shows small positive dots, but no diffuse signal in the epithelium, which could represent a later developmental event. no positive signal was observed by ish using lung materials of fetuses from litters sacrificed on gd, which is also compatible with a transition state. lpl protein was found in capillary-like structures on gd, gd, and gd. lpl mrna was mainly found in epithelial cells of the distal epithelium on gd. this mrna was absent of the proximal epithelium on gd except for a few cells that may present barely detectable signals. on gd, lpl mrna was mainly found in the mesenchyme and the distal epithelium as for apoc-ii mrna. on gd, no signal was obtained by ish for lpl using lung tissues of fetuses from litters. in order to confirm that the lpl-positive signal corresponds to capillaries and not to α-smooth muscle actin -positive structures, an anti-platelet endothelial cell adhesion molecule- antibody was used on gd -lung tissue sections to stain capillaries. results show that staining of capillary network is very similar to the positive signals identified as capillary-like structures using an anti-lpl antibody. in contrast, the use of an anti-α-sma antibody leads to a different staining profile. apoc-ii and lpl mrnas were observed in smooth muscles surrounding large arteries. lpl protein was found in smooth muscles of arteries, but signal intensities were lower than those found in adjacent capillaries. no ihc signal was found in smooth muscles for apoc-ii. in most cases, it was difficult to determine whether endothelial cells of large vessels were stained because of the proximity to positive structures. nevertheless, endothelial cells positive for lpl by ihc and others negative for apoc-ii by ish and ihc were also found on gd. apoc-ii and lpl mrna levels during the saccular stage and the first segment of the alveolar stage apoc-ii and lpl qpcr data were recently published for samples from gd to gd, but not for further developmental times. here, qpcr analysis of these two genes is presented between gd and pn using male and female pools of various litters. levels of apoc-ii mrna showed more variations from sample to sample than those of lpl mrna. there was no statistically significant sex difference for the two genes, even though higher values were observed for males for apoc-ii mrna on pn in four of the five analyzed litters. a statistically significant difference according to developmental time was observed only for lpl mrna. mrna and protein localization of lpl and apoc-ii during the saccular and the beginning of the alveolar stages of lung development similarities were found between apoc-ii and lpl expression patterns during the perinatal period. both genes were expressed in the distal epithelium short time before birth, but not after birth. in contrast, the proximal epithelium was negative on gd, showed a weak positive signal on gd and, presented a marked increase in intensity soon after birth, and was negative on pn for both genes. using fetuses on gd and neonates on pn we confirmed that the switch from the distal to the proximal epithelium correlated with birth, not with sex. apoc-ii and lpl mrnas were also found in a few scattered distal epithelial cells after birth. in addition, positive signals by ish were observed in association with blood vessels before birth but not after birth for both genes. on pn thus at junction between saccularization and alveolarization, apoc-ii and lpl mrna were found in alveolar walls, including newly-formed septa. albeit similarities were observed in the expression profiles of their encoding genes, apoc-ii and lpl proteins did not accumulate in the same structures. interestingly, apoc-ii was observed in secretory granule-like structures until soon after birth, but positive signal had disappeared one day later. in fact, between gd and pn apoc-ii-positive secretory granules were mainly observed in distal epithelia with no or small lumina, but not with large lumina. such a discrepancy was less evident for apoc-ii mrna for which some distal epithelia with large lumina were also positive. this strongly suggests that apoc-ii protein is synthesized and retained within the epithelium in secretory granules until the lumina reaches a certain diameter, and that it is then secreted. we did not observe any sex difference or developmental delay in our experiment in that the three males and the three females analyzed on pn presented secretory granules, while the six other animals studied on pn did not show any secretory granule. in all of the experiments, neonates sacrificed on pn were killed between a few minutes and four hours after the birth of the first neonate. the apoc-ii protein was still undetectable on pn, while secretory granules were found in the epithelial cells of the respiratory bronchioles on pn near the basal membrane, close to the mesenchyme. only a few secretory granules were still positive on pn apoc-ii-positive capillaries, as those shown on figure 5o, were only observed in one third of the subjects analyzed on pn in fact, after its secretion, apoc-ii protein may be difficult to detect depending of the nature of its association with the tissue. the situation was clearly different for lpl. the protein was found in capillaries from pn to pn for all the analyzed neonates as well as before birth. lpl positive signals were also found in cells of the respiratory bronchiole from gd to pn, but not on gd and pn in this epithelium, signal intensity showed variation from cell to cell. in contrast, no protein was detected in most proximal epithelium. cells localized in the alveolar space on pn were positive for apoc-ii and lpl by both ish and ihc with no sex difference. based on their localization and morphology, these cells were most probably macrophages. apoc-ii and lpl proteins were also found in macrophages on pn. disaturated phosphatidylcholine determination disaturated phosphatidylcholine is quantitatively the most important lipid of surfactant and an increase in lung dspc content was observed after the surge of surfactant synthesis. here, relative dspc levels were determined in lungs from balb/c fetal mice from gd to following a one hour mating window. a marked statistically significant increase was observed between gd and gd. discussion surfactant is of the first importance in the distal epithelium to prevent alveolar collapse. our results show that the lpl-related molecular machinery is regulated temporally and geographically in the developing lung in correlation with the need in surfactant lipid synthesis. major changes in sites of apoc-ii and lpl mrna are summarized in table changes in protein localization were mainly observed for apoc-ii and are summarized in table our results show that the tissue needing surfactant governs itself lpl and apoc-ii gene expression and apoc-ii secretion during the critical period. thus, the lpl machinery is subjected to a complex regulation during the canalicular and the saccular stages of lung development. the need of lipids for surfactant synthesis is particularly important during the period between the surge of surfactant synthesis and birth. this can explain why the distal epithelium of the lung contributes itself to lpl and apoc-ii synthesis, which must maximizes fatty acid recruitment during this period. our results are compatible with those of ryan et al showing that maternal loading with vldl stimulates fetal surfactant synthesis. * apoc-ii was observed in capillaries only in one third of the analyzed subjects on pn 10 one major observation about apoc-ii protein is that it accumulated in secretory granules within several distal epithelia from gd to a developmental time point soon after birth. thus, a temporal and geographic correlation exists between the appearance of lamellar bodies as reported in the literature and apoc-ii accumulation in secretory granules presented in figure in contrast to apoc-ii, lpl protein was obviously secreted out of the producing cells and accumulated in capillaries. accordingly, a rapid release of 10-15% of total tissue lpl was observed following infusion of heparin into isolated perfused adult rat lungs. the presence of lpl in capillaries in the fetal lung is compatible with its recognized function in recruitment of fatty acids from lipoprotein-associated triglycerides. in contrast to lpl, apoc-ii was observed in its producing cells within secretory granules. this suggests that apoc-ii secretion is controlled, but not at all developmental time points. for example, no secretory granule is observed on gd indicating that there is no apoc-ii retention within the producing cells. a transition state is observed on gd with small secretory granules, which must correspond to the retention of newly formed apoc-ii molecules. from gd to pn distal epithelia with no or small lumina containing apoc-ii-positive secretory granules as well as distal epithelia with large lumina negative for apoc-ii were observed. as explained in figure these observations suggest that apoc-ii is secreted by distal epithelia reaching a specific level of development, which stimulates local lpl activity for fatty acid recruitment for surfactant synthesis. in contrast to lpl, apoc-ii was not reported to bind to the endothelium and consequently, most of the apoc-ii molecules secreted by the epithelia would be washed off of the slide during the ihc procedure so that the presence of apoc-ii in capillaries could be difficult to demonstrate. the fact that apoc-ii is produced by the lung and not only provided by plasma for activation of lpl in capillaries could be explained by the great need for fatty acids for surfactant production and, consequently, the very high amount of lpl molecules necessitating apoc-ii for their activation. a noticeable increase in surfactant lipid content was observed between gd and. knowing that surfactant lipids cannot be detected immediately when they start to be produced but have to accumulate over the detection limit before an increase be observed, our results are compatible with the literature showing the appearance of lamellar bodies on gd. moreover, the marked increase in dspc levels coincides with the presence of distal epithelium with large lumina without secretory granules, thus after the beginning of apoc-ii secretion for optimization of surfactant lipid production based on our model. in lpl expression was found in lipofibroblasts isolated from rat developing lungs. more specifically, fold and fold increases in lpl mrna levels were observed by northern blot analysis in isolated lipofibroblasts and whole lungs, respectively, between postnatal days - and + in our experiments, not only did we fail to reproduce this rise at birth in whole mouse lungs by quantitative real time pcr, but lpl mrna accumulation sites changed from distal to proximal epithelium at birth with no or only a few positive signal in the mesenchyme after birth. furthermore, the ihc signal we obtained for lpl was close to the background in the mesenchyme during the perinatal period. of course, it is not excluded that lpl expression in lipofibroblasts be very low and under the limit of detection in our assays, but obviously, our data indicate that this cell population is not a major site of lpl mrna and protein accumulation. the results of chen et al are also incompatible with other studies reporting many years ago that pulmonary lpl activity in growing rats peaks one day before birth and decreases at birth. the decrease at birth may correspond to the switch we observed in the site of lpl gene expression in the mouse. in fact, the timing of lpl activity in the rats, the temporal and cell specific regulations of lpl gene expression we observed in the mouse and the presence of lpl in capillaries underscore a role in recruitment of lipids for the surge of surfactant synthesis. such a role has been already proposed based on the presence of vldl in the fetal circulation at days' gestation in the rat when the peak in lpl activity occurs. lpl proteins were also found in other sites in the developing lung as in respiratory bronchioles during saccularization and in macrophages from the beginning of alveolarization. as reported for rats, an increase in lpl activity was observed after the first week of life following the decrease at birth. this observation is compatible with the increase we observed in lpl mrna from pn to and may be explained by apparition of intra-alveolar macrophages, which express lpl as reported here and in the literature. our data cannot support or exclude lpl expression by lipofibroblasts at this developmental time. no sex difference was observed in sites of apoc-ii and lpl gene expression and protein accumulation for any of the analyzed time point. no sex difference in signal intensity was observed, although ish and ihc are not reliable quantitative tools. qpcr analysis did not reveal any significant sex difference when all the population was considered, but some differences from sample to sample prevented us to conclude at the absence of any sex difference at specific time points, as at pn when higher levels of apoc-ii mrna were observed more frequently in males. the sex difference reported recently in apoc-ii mrna levels in favor of females in developmental times preceding gd is not ruled out by the present work. this statistically significant sex difference in apoc-ii mrna suggests that a significant proportion of females would start accumulating apoc-ii earlier than males. hypertriglyceridemia is observed in both lpl and apoc-ii deficiencies, while there is no evidence that these deficiencies may be associated with a high rate of mortality related to respiratory distress syndrome of the neonate in the human. the situation is different in lpl knockout mice, which present a high rate of neonatal death caused by respiratory problems. however, the cause of death was engorgement of capillaries by chylomicrons leading to cyanosis, not surfactant insufficiency. therefore, apoc-ii and lpl seem not essential for obtaining minimal surfactant levels following birth at term. this situation is not surprising knowing that lpl activity is only one of the two sources of fatty acids, the remaining source being de novo synthesis through fatty acid synthase. as it is frequently observed for essential mechanisms/metabolisms, a redundancy is observed here in the source of fatty acids for surfactant synthesis. interestingly, in mice deficient in adipose tissue lpl, fat mass was preserved by large increases in endogenous fatty acid biosynthesis, which compensated for the loss of lpl activity. a similar situation should occur in the developing lung of lpl- and apoc-ii-deficient subjects. in fact, the importance of the work presented here lies in the fact that preterm birth frequently leads to surfactant insufficiency and therefore, apoc-ii/lpl may become an interesting pharmaceutical target in that context.  we have shown that sites of apoc-ii as well as lpl mrna and protein accumulation are regulated during the canalicular and the saccular stages of lung development. these changes show a complex regulation of the lpl molecular machinery during an important period for the adaptation of the lung to gas exchange. from this work, apoc-ii appears to be an interesting potential pharmaceutical target for treatment and/or prevention of respiratory distress syndrome of the neonate. 
the present study investigated whether the sites of apoc-ii and lpl mrna and protein accumulation are regulated in the mouse lung between gestation day and postnatal day the major sites of apoc-ii and lpl gene expression changed over time and were found mainly in the distal epithelium at the end of gestation but not after birth. in the present study, we will answer the following questions: does apoc-ii protein localization change according to the stage of lung development, thus according to the need in surfactant? do the sites of apoc-ii and lpl gene expression change according to the stage of lung development and to protein localization? a noticeable increase in surfactant lipid content was measured before the end of gestation day which correlates temporally with the presence of apoc-ii in secretory granules in distal epithelium with no or small lumina but not with large lumina. we propose a model where apoc-ii is retained in secretory granules in distal epithelial cells until the lumina reaches a minimum size, and is then secreted when the rate of surfactant production becomes optimal. recently, we showed expression of apolipoprotein c-ii, the essential cofactor of lipoprotein lipase, in the fetal mouse lung and found the protein on the day of the surge of surfactant synthesis in secretory granule-like structures in the distal epithelium. accumulation of apoc-ii in secretory granule-like structures was not systematically observed, but was found in the distal epithelium only at the end of gestation and soon after birth, mainly in epithelia with no or small lumina. are lpl molecules translocated to the luminal surface of capillaries? this study demonstrates that apoc-ii and lpl mrnas correlate temporally and geographically with surfactant lipid synthesis in preparation for birth and suggests that fatty acid recruitment from the circulation by apoc-ii-activated lpl is regionally modulated by apoc-ii secretion. fatty acids are precursors in the synthesis of surfactant phospholipids. lpl was detected in capillaries at all the developmental times studied.
 fatty acids are precursors in the synthesis of surfactant phospholipids. recently, we showed expression of apolipoprotein c-ii, the essential cofactor of lipoprotein lipase, in the fetal mouse lung and found the protein on the day of the surge of surfactant synthesis in secretory granule-like structures in the distal epithelium. in the present study, we will answer the following questions: does apoc-ii protein localization change according to the stage of lung development, thus according to the need in surfactant? are lpl molecules translocated to the luminal surface of capillaries? do the sites of apoc-ii and lpl gene expression change according to the stage of lung development and to protein localization?  the present study investigated whether the sites of apoc-ii and lpl mrna and protein accumulation are regulated in the mouse lung between gestation day and postnatal day the major sites of apoc-ii and lpl gene expression changed over time and were found mainly in the distal epithelium at the end of gestation but not after birth. accumulation of apoc-ii in secretory granule-like structures was not systematically observed, but was found in the distal epithelium only at the end of gestation and soon after birth, mainly in epithelia with no or small lumina. a noticeable increase in surfactant lipid content was measured before the end of gestation day which correlates temporally with the presence of apoc-ii in secretory granules in distal epithelium with no or small lumina but not with large lumina. lpl was detected in capillaries at all the developmental times studied.  this study demonstrates that apoc-ii and lpl mrnas correlate temporally and geographically with surfactant lipid synthesis in preparation for birth and suggests that fatty acid recruitment from the circulation by apoc-ii-activated lpl is regionally modulated by apoc-ii secretion. we propose a model where apoc-ii is retained in secretory granules in distal epithelial cells until the lumina reaches a minimum size, and is then secreted when the rate of surfactant production becomes optimal. 