Saccular Phase Research Articles

Relationship between the sonographic foetal lung-to-liver ratio of mean grey level and progesterone concentration in canine late gestation

A study was conducted to perform the quantitative analysis of canine foetal lung and liver sonographic images in the late term of gestation (58-67 days) and to evaluate its association with serum progesterone concentration. Thirty-two pregnant bitches were selected for the study. Lung development of foetus occurs in three different stages viz the pseudo glandular (40-48), canalicular (49-56 d) and saccular phase (57-63 d) of pregnancy. Lungs of the foetus at the time of parturition are in saccular phase of development and the alveolar phase occurs in the neonatal period. In the present study, sonographic changes occurring in the lung during the saccular phase of foetal lung development was evaluated. Mean grey level of the foetal lung and liver sonographic images were estimated using dedicated image analysis software Image J. The lung-to-liver ratio (LLR) of mean grey level (MGL) was recorded. Corresponding progesterone concentrations were also estimated for each animal. Correlation statistical analysis revealed a significant correlation between LLR of MGL and progesterone concentration in the animals selected for the study. LLR of MGL is a prognostic indicator of foetal survival and since LLR of MGL and progesterone are positively correlated, it can be used as an indicator of foetal maturity in place of progesterone concentration. Keywords: Lung-to-liver ratio of mean grey level, ultrasonography, progesterone

Prenatal FGFR2 Signaling via PI3K/AKT Specifies the PDGFRA+ Myofibroblast.

It is well known that FGFR2 (fibroblast growth factor receptor 2) signaling is critical for proper lung development. Recent studies demonstrate that epithelial FGFR2 signaling during the saccular phase of lung development (sacculation) regulates alveolar type 1 (AT1) and AT2 cell differentiation. During sacculation, PDGFRA (platelet-derived growth factor receptor-α)-positive lung fibroblasts exist as three functional subtypes: contractile myofibroblasts, extracellular matrix-producing matrix fibroblasts, and lipofibroblasts. All three subtypes are required during alveolarization to establish a niche that supports AT2 epithelial cell self-renewal and AT1 epithelial cell differentiation. FGFR2 signaling directs myofibroblast differentiation in PDGFRA+ fibroblasts during alveolar reseptation after pneumonectomy. However, it remains unknown if FGFR2 signaling regulates PDGFRA+ myo-, matrix, or lipofibroblast differentiation during sacculation. In this study, FGFR2 signaling was inhibited by temporal expression of a secreted dominant-negative FGFR2b (dnFGFR2) by AT2 cells from embryonic day (E) 16.5 to E18.5. Fibroblast and epithelial differentiation were analyzed at E18.5 and postnatal days 7 and 21. At all time points, the number of myofibroblasts was reduced and the number of lipo-/matrix fibroblasts was increased. AT2 cells are increased and AT1 cells are reduced postnatally, but not at E18.5. Similarly, in organoids made with PDGFRA+ fibroblasts from dnFGFR2 lungs, increased AT2 cells and reduced AT1 cells were observed. In vitro treatment of primary wild-type E16.5 adherent saccular lung fibroblasts with recombinant dnFGFR2b/c resulted in reduced myofibroblast contraction. Treatment with the PI3K/AKT activator 740 Y-P rescued the lack of myofibroblast differentiation caused by dnFGFR2b/2c. Moreover, treatment with the PI3K/AKT activator 740 Y-P rescued myofibroblast differentiation in E18.5 fibroblasts isolated from dnFGFR2 lungs.

Time-resolved transcriptomic profiling of the developing rabbit’s lungs: impact of premature birth and implications for modelling bronchopulmonary dysplasia

BackgroundPremature birth, perinatal inflammation, and life-saving therapies such as postnatal oxygen and mechanical ventilation are strongly associated with the development of bronchopulmonary dysplasia (BPD); these risk factors, alone or combined, cause lung inflammation and alter programmed molecular patterns of normal lung development. The current knowledge on the molecular regulation of lung development mainly derives from mechanistic studies conducted in newborn rodents exposed to postnatal hyperoxia, which have been proven useful but have some limitations.MethodsHere, we used the rabbit model of BPD as a cost-effective alternative model that mirrors human lung development and, in addition, enables investigating the impact of premature birth per se on the pathophysiology of BPD without further perinatal insults (e.g., hyperoxia, LPS-induced inflammation). First, we characterized the rabbit’s normal lung development along the distinct stages (i.e., pseudoglandular, canalicular, saccular, and alveolar phases) using histological, transcriptomic and proteomic analyses. Then, the impact of premature birth was investigated, comparing the sequential transcriptomic profiles of preterm rabbits obtained at different time intervals during their first week of postnatal life with those from age-matched term pups.ResultsHistological findings showed stage-specific morphological features of the developing rabbit’s lung and validated the selected time intervals for the transcriptomic profiling. Cell cycle and embryo development, oxidative phosphorylation, and WNT signaling, among others, showed high gene expression in the pseudoglandular phase. Autophagy, epithelial morphogenesis, response to transforming growth factor β, angiogenesis, epithelium/endothelial cells development, and epithelium/endothelial cells migration pathways appeared upregulated from the 28th day of gestation (early saccular phase), which represents the starting point of the premature rabbit model. Premature birth caused a significant dysregulation of the inflammatory response. TNF-responsive, NF-κB regulated genes were significantly upregulated at premature delivery and triggered downstream inflammatory pathways such as leukocyte activation and cytokine signaling, which persisted upregulated during the first week of life. Preterm birth also dysregulated relevant pathways for normal lung development, such as blood vessel morphogenesis and epithelial-mesenchymal transition.ConclusionThese findings establish the 28-day gestation premature rabbit as a suitable model for mechanistic and pharmacological studies in the context of BPD.

Wnt5a Promotes AT1 and Represses AT2 Lineage-Specific Gene Expression in a Cell-Context-Dependent Manner.

Lung maturation is not limited to proper structural development but also includes differentiation and functionality of various highly specialized alveolar cell types. Alveolar type 1 (AT1s) cells occupy nearly 95% of the alveolar surface and are critical for establishing efficient gas exchange in the mature lung. AT1 cells arise from progenitors specified during the embryonic stage as well as alveolar epithelial progenitors expressing surfactant protein C (Sftpcpos cells) during postnatal and adult stages. Previously, we found that Wnt5a, a non-canonical Wnt ligand, is required for differentiation of AT1 cells during the saccular phase of lung development. To further investigate the role of Wnt5a in AT1 cell differentiation, we generated and characterized a conditional Wnt5a gain-of-function mouse model. Neonatal Wnt5a gain-of-function disrupted alveologenesis through inhibition of cell proliferation. In this setting Wnt5a downregulated β-catenin-dependent canonical Wnt signaling, repressed AT2 (anti-AT2) and promoted AT1 (pro-AT1) lineage-specific gene expression. In addition, we identified 2 subpopulations of Sftpchigh and Sftpclow alveolar epithelial cells. In Sftpclow cells, Wnt5a exhibits pro-AT1 and anti-AT2 effects, concurrent with inhibition of canonical Wnt signaling. Interestingly, in the Sftpchigh subpopulation, although increasing AT1 lineage-specific gene expression, Wnt5a gain-of-function did not change AT2 gene expression, nor inhibit canonical Wnt signaling. Using primary epithelial cells isolated from human fetal lungs, we demonstrate that this property of Wnt5a is evolutionarily conserved. Wnt5a therefore serves as a selective regulator that ensures proper AT1/AT2 balance in the developing lung.

Microscopic Studies Of The Foetal Lung Development In The OneHumped Camel (Camelus dromedarius) Obtained From Kano Abattoir

Eighty – seven foetal lung samples from camels (Camelus dromedarius) were collected at Kano abattoir to study lung development. Four phases of the lung development were observed as follows; pseudo-glandular phase was between 10.5 cm to 38.2 cm crown-rump length, with endodermal buds and prominent mesenchymal cells. Canalicular phase was observed between 38.7 cm to 62.2 cm crownrump length and was characterized by thinning of the endodermal buds and invasion of capillaries. Saccular phase was noticed at 65.6 cm to 82.6 cm crown-rump length when thick intercellular septa were noticed. Alveolar phase was noticed between 85.9 cm to 121 cm crown-rump length when primitive alveolar was noticed. This study has established phases of lung development in the camel and serves as baseline information for scientific knowledge.
 Keywords: Foetal lung, camel (Camelus dromedarius), crown-rump length, lung development and Kano abattoir.

Maternal exposure to PM2.5 during pregnancy and asthma risk in early childhood: consideration of phases of fetal lung development.

Increasingly studies suggest prenatal exposure to air pollution may increase risk of childhood asthma. Few studies have investigated exposure during specific fetal pulmonary developmental windows. To assess associations between prenatal fine particulate matter exposure and asthma at age 4. This study included mother-child dyads from two pregnancy cohorts-CANDLE and TIDES-within the ECHO-PATHWAYS consortium (births in 2007-2013). Three child asthma outcomes were parent-reported: ever asthma, current asthma, and current wheeze. Fine particulate matter (PM2.5) exposures during the pseudoglandular (5-16 weeks gestation), canalicular (16-24 weeks gestation), saccular (24-36 weeks gestation), and alveolar (36+ weeks gestation) phases of fetal lung development were estimated using a national spatiotemporal model. We estimated associations with Poisson regression with robust standard errors, and adjusted for child, maternal, and neighborhood factors. Children (n=1469) were on average 4.3 (standard deviation 0.5) years old, 49% were male, and 11.7% had ever asthma; 46% of women identified as black and 53% had at least a college/technical school degree. A 2 μg/m3 higher PM2.5 exposure during the saccular phase was associated with 1.29 times higher risk of ever asthma (95% CI: 1.06-1.58). A similar association was observed with current asthma (RR 1.27, 95% CI: 1.04-1.54), but not current wheeze (RR 1.11, 95% CI: 0.92-1.33). Effect estimates for associations during other developmental windows had confidence intervals that included the null. Later phases of prenatal lung development may be particularly sensitive to the developmental toxicity of PM2.5.

Circular RNAs are abundant and dynamically expressed during the embryonic lung development of C57BL/6 mice

Circular RNAs (circRNAs), a novel type of endogenous RNAs, can function as microRNA (miRNA) sponges capable of regulating gene transcription, binding to RNA-associated proteins, and even encoding proteins. CircRNAs are involved in various cell behaviors, such as proliferation and apoptosis. The mouse model has also been demonstrated to be similar to that of humans in many studies. To explore the profile of circRNAs during embryonic lung development and their potential functions in lung development-related diseases, mouse embryos at the pseudoglandular phase, canalicular phase, saccular phase, and alveolar phase were collected. High-throughput sequencing was then used to identify a total of 1,735 circRNAs (junction reads ≥5 and p < 0.05). It is well known that the functions of circRNAs are related to host genes. In our study, bioinformatics analysis indicated that the screened host genes were closely associated with lung development and included the Hippo signaling pathway, PI3K-Akt signaling pathways, and TGF-β signaling pathways. Moreover, miRNA sponges are another mechanism involved in lung development. Therefore, we predicted many miRNAs binding to circRNAs, such as miR-17 and miR-20, using the TargetScan and miRanda databases. Previously, miRNAs were proven to be necessary for lung development. The peak expression of circRNAs is distributed at different time points, suggesting their involvement in different stages of embryonic mouse lung development.

WNT5a-ROR Signaling Is Essential for Alveologenesis.

WNT5a is a mainly “non-canonical” WNT ligand whose dysregulation is observed in lung diseases such as idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and asthma. Germline deletion of Wnt5a disrupts embryonic lung development. However, the temporal-specific function of WNT5a remains unknown. In this study, we generated a conditional loss-of-function mouse model (Wnt5aCAG) and examined the specific role of Wnt5a during the saccular and alveolar phases of lung development. The lack of Wnt5a in the saccular phase blocked distal airway expansion and attenuated differentiation of endothelial and alveolar epithelial type I (AT1) cells and myofibroblasts. Postnatal Wnt5a inactivation disrupted alveologenesis, producing a phenotype resembling human bronchopulmonary dysplasia (BPD). Mutant lungs showed hypoalveolization, but endothelial and epithelial differentiation was unaffected. The major impact of Wnt5a inactivation on alveologenesis was on myofibroblast differentiation and migration, with reduced expression of key regulatory genes. These findings were validated in vitro using isolated lung fibroblasts. Conditional inactivation of the WNT5a receptors Ror1 and Ror2 in alveolar myofibroblasts recapitulated the Wnt5aCAG phenotype, demonstrating that myofibroblast defects are the major cause of arrested alveologenesis in Wnt5aCAG lungs. Finally, we show that WNT5a is reduced in human BPD lung samples, indicating the clinical relevance and potential role for WNT5a in pathogenesis of BPD.

Prenatal air pollution and early childhood asthma

PDS 68: Outdoor air pollution, mortality and morbidity, Exhibition Hall (PDS), Ground floor, August 26, 2019, 10:30 AM - 12:00 PM Background: Prior studies suggest that prenatal air pollution may perturb normal fetal lung development and contribute to risk of child asthma, but few have had opportunity to investigate developmental windows of susceptibility or have been limited in exposure assessment or covariate adjustment. Methods: Our study population included participants in two pregnancy cohorts within the ECHO-PATHWAYS consortium, CANDLE (births in 2007-2011) in Memphis, TN and TIDES (births in 2011-2013) in Seattle, WA; San Francisco, CA; and Minneapolis, MN, who had term births (37+ weeks). Child asthma was defined based on parent-report of ever asthma. Exposure to ambient fine particulate matter (PM2.5) during prenatal phases of fetal lung development was estimated using a spatiotemporal model. We used Poisson regression and adjusted for city, child sex, year and season of birth, maternal race, education, asthma, age at delivery, prior live births, pre-pregnancy body mass index, prenatal smoking, child’s postnatal secondhand smoke exposure, pets in the child’s home, and neighborhood-level socioeconomic status. Results: Children (n=1334) were on average 4.3 years old (standard deviation [SD] 0.5), 49% male, and 11.6% had ever asthma; 46% of mothers were black and 54% had a college/technical school degree. City-specific mean PM2.5 exposures averaged across the prenatal period ranged from 5.2 µg/m3 (SD 0.9) to 10.6 µg/m3 (SD 1.0). A 2 µg/m3 higher PM2.5 exposure during the saccular phase (week 24 to week 36 of pregnancy) was associated with 28% higher risk of asthma (95% confidence interval [CI]: 1.00, 1.64; p=0.055). This effect was larger in boys than in girls, though interaction was not statistically significant. Higher average exposure across all of pregnancy was not associated with asthma (risk ratio 1.06, 95% CI: 0.84, 1.35). Conclusions: Preliminary results suggest that the later phases of prenatal lung development may be particularly sensitive to the developmental toxicity of PM2.5.

Consequences of early postnatal lipopolysaccharide exposure on developing lungs in mice.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of infants that is characterized by interrupted lung development. Postnatal sepsis causes BPD, yet the contributory mechanisms are unclear. To address this gap, studies have used lipopolysaccharide (LPS) during the alveolar phase of lung development. However, the lungs of infants who develop BPD are still in the saccular phase of development, and the effects of LPS during this phase are poorly characterized. We hypothesized that chronic LPS exposure during the saccular phase disrupts lung development by mechanisms that promote inflammation and prevent optimal lung development and repair. Wild-type C57BL6J mice were intraperitoneally administered 3, 6, or 10 mg/kg of LPS or a vehicle once daily on postnatal days (PNDs) 3-5. The lungs were collected for proteomic and genomic analyses and flow cytometric detection on PND6. The impact of LPS on lung development, cell proliferation, and apoptosis was determined on PND7. Finally, we determined differences in the LPS effects between the saccular and alveolar lungs. LPS decreased the survival and growth rate and lung development in a dose-dependent manner. These effects were associated with a decreased expression of proteins regulating cell proliferation and differentiation and increased expression of those mediating inflammation. While the lung macrophage population of LPS-treated mice increased, the T-regulatory cell population decreased. Furthermore, LPS-induced inflammatory and apoptotic response and interruption of cell proliferation and alveolarization was greater in alveolar than in saccular lungs. Collectively, the data support our hypothesis and reveal several potential therapeutic targets for sepsis-mediated BPD in infants.

Estimation of fetal lung development using quantitative analysis of ultrasonographic images in normal canine pregnancy

We investigated the quantitative analysis of sonographic images to predict fetal lung maturity of the canine foetus in normal pregnancy. Twelve bitches were recruited in the present study. Serial ultrasonographic exams were performed at three pre-determined time periods corresponding to the pseudoglandular (40–48 days of pregnancy), canalicular (49–56 days of pregnancy) and saccular phase (57–63 days of pregnancy) of lung development. Mean grey level (MGL) and the standard deviation of the histogram (SDH) of fetal lung and liver sonographic images were measured with dedicated software. The lung-to-liver ratio (LLR) for both parameters was also calculated. Measurements were taken on the two caudal-most foetuses and then averaged. SDH did not show any statistically significant difference between the three time periods in the lungs or in the liver. MGL measured in the lungs significantly increased in the first period and reached a plateau during the last two periods. Liver echogenicity was constant during the first two periods and significantly increased during the last week of gestation. The LLR of MGL significantly decreased during the last week of pregnancy. The LLR was a very good test to detect fetal lung maturity (area under the receiver operator curve (AUROC) = 0.875); using a cut-off value of LLR < 1.541, sensitivity was 83.33% and specificity was 83.33%, positive likelihood ratio = 5. LLR of MGL is an accurate test to estimate lung development in normal canine pregnancies.

Co-expression of TTF-1 and neuroendocrine markers in the human fetal lung and pulmonary neuroendocrine tumors

The expression pattern of thyroid transcription factor 1 (TTF-1) and neuroendocrine markers, neuron cell adhesion molecule (NCAM; CD56), chromogranin A (CgA) and synaptophysin (Syp), of different lung cell lineages was histologically analyzed in 15 normal human fetal lungs and 12 neuroendocrine tumors (NETs) using immunohistochemical methods. During pseudoglandular phase strong nuclear TTF-1 staining was detected in the columnar nonciliated epithelial cells, while NCAM, CgA and Syp had a moderate expression in the proximal airways and mild expression in the distal airways. Neuroendocrine cells (NECs) in proximal lung airway were co-localizing TTF-1 and other neuroendocrine markers while neuroendocrine bodies (NEBs) exhibit only staining with NCAM and Syp. In the canalicular phase TTF-1 nuclear staining was expressed only in several epithelial cells in proximal airways, while budding airways epithelium showed strong TTF-1 expression. Expression of NCAM, CgA and Syp in this phase equals the one in pseudoglandular phase. NEBs cells were co-localizing TTF-1 and NCAM in proximal airways and few NECs in distal airway were co-localizing TTF-1 and Syp. TTF-1 staining in the saccular phase was limited to subsets of epithelial cells in the proximal airways with stronger positivity in the distal airways. NCAM expression is moderate only in proximal airways, while Syp and CgA show mild expression in proximal and distal airways. NECs were co-localizing TTF-1 and NCAM in proximal lung airway. With regard to NECs, all small cell lung cancer (SCLC) cells had strong TTF-1, NCAM, Syp and CgA positivity and TTF-1 co-localized with other neuroendocrine markers. All pulmonary typical carcinoids were TTF-1 negative, while pulmonary atypical carcinoids were focal positive for TTF-1 and some neoplastic cells co-localized TTF-1 with neuroendocrine markers.Our results indicate that TTF-1 expression in NECs suggests a possible role in their normal development and differentiation. Our results also indicate that possible cell of origin for poorly differentiated SCLC and some atypical carcinoid could be a progenitor cell in neuroendocrine lineage while in typical carcinoids possible cell of origin is localized in terminally differentiated NECs.

Progenitors of secondary crest myofibroblasts are developmentally committed in early lung mesoderm.

Development of the mammalian lung is predicated on cross-communications between two highly interactive tissues, the endodermally derived epithelium and the mesodermally derived pulmonary mesenchyme. While much attention has been paid for the lung epithelium, the pulmonary mesenchyme, partly due to lack of specific tractable markers remains under-investigated. The lung mesenchyme is derived from the lateral plate mesoderm and is the principal recipient of Hedgehog (Hh) signaling, a morphogenetic network that regulates multiple aspects of embryonic development. Using the Hh-responsive Gli1-cre(ERT2) mouse line, we identified the mesodermal targets of Hh signaling at various time points during embryonic and postnatal lung development. Cell lineage analysis showed these cells serve as progenitors to contribute to multiple lineages of mesodermally derived differentiated cell types that include parenchymal or interstitial myofibroblasts, peribronchial and perivascular smooth muscle as well as rare populations of cells within the mesothelium. Most importantly, Gli1-cre(ERT2) identified the progenitors of secondary crest myofibroblasts, a hitherto intractable cell type that plays a key role in alveolar formation, a vital process about which little is currently known. Transcriptome analysis of Hh-targeted progenitor cells transitioning from the pseudoglandular to the saccular phase of lung development revealed important modulations of key signaling pathways. Among these, there was significant downregulation of canonical WNT signaling. Ectopic stabilization of β-catenin via inactivation of Apc by Gli1-cre(ERT2) expanded the Hh-targeted progenitor pools, which caused the formation of fibroblastic masses within the lung parenchyma. The Gli1-cre(ERT2) mouse line represents a novel tool in the analysis of mesenchymal cell biology and alveolar formation during lung development.

A preterm pig model of lung immaturity and spontaneous infant respiratory distress syndrome.

Respiratory distress syndrome (RDS) and bronchopulmonary dysplasia remain the leading causes of preterm infant morbidity, mortality, and lifelong disability. Research to improve outcomes requires translational large animal models for RDS. Preterm pigs delivered by caesarian section at gestation days (GD) 98, 100, 102, and 104 were provided 24 h of neonatal intensive care, monitoring (pulse oximetry, blood gases, serum biomarkers, radiography), and nutritional support, with or without intubation and mechanical ventilation (MV; pressure control ventilation with volume guarantee). Spontaneous development of RDS and mortality without MV are inversely related with GD at delivery and correspond with inadequacy of tidal volume and gas exchange. GD 98 and 100 pigs have consolidated lungs, immature alveolar architecture, and minimal surfactant protein-B expression, and MV is essential at GD 98. Although GD 102 pigs had some alveoli lined by pneumocytes and surfactant was released in response to MV, blood gases and radiography revealed limited recruitment 1-2 h after delivery, and mortality at 24 h was 66% (35/53) with supplemental oxygen provided by a mask and 69% (9/13) with bubble continuous positive airway pressure (8-9 cmH2O). The lungs at GD 104 had higher densities of thin-walled alveoli that secreted surfactant, and MV was not essential. Between GD 98 and 102, preterm pigs have ventilation inadequacies and risks of RDS that mimic those of preterm infants born during the saccular phase of lung development, are compatible with standards of neonatal intensive care, and are alternative to fetal nonhuman primates and lambs.

Functional assessment of hyperoxia-induced lung injury after preterm birth in the rabbit

The objective of this study was to document early neonatal (7 days) pulmonary outcome in the rabbit model for preterm birth and hyperoxia-induced lung injury. Preterm pups were delivered at 28 days (term = 31 days; early saccular phase of lung development) by cesarean section, housed in an incubator, and gavage fed for 7 days. Pups were divided into the following groups: 1) normoxia (21% O2; normoxia group) and 2) and hyperoxia (>95% O2; hyperoxia group). Controls were pups born at term who were housed in normoxic conditions (control group). Outcome measures were survival, pulmonary function tests using the whole body plethysmograph and forced oscillation technique, and lung morphometry. There was a significant difference in survival of preterm pups whether they were exposed to normoxia (83.3%) or hyperoxia (55.9%). Hyperoxic exposure was associated with increased tissue damping and elasticity and decreased static compliance compared with normoxic controls (P < 0.01). Morphometry revealed an increased linear intercept and increased mean wall transection length, which translates to larger alveoli with septal thickening in hyperoxia compared with normoxia (P < 0.01). In conclusion, the current experimental hyperoxic conditions to which preterm pups are exposed induce the typical clinical features of bronchopulmonary dysplasia. This model will be used to study novel preventive or therapeutic interventions.

Abrogation of Eya1/Six1 disrupts the saccular phase of lung morphogenesis and causes remodeling

The Eya1 gene encodes a transcriptional co-activator that acts with Six1 to control the development of different organs. However, Six1-Eya1 interactions and functional roles in mesenchymal cell proliferation and differentiation as well as alveolarization during the saccular stage of lung development are still unknown. Herein, we provide the first evidence that Six1 and Eya1 act together to regulate mesenchymal development as well as alveolarization during the saccular phase of lung morphogenesis. Deletion of either or both Six1 and Eya1 genes results in a severe saccular phenotype, including defects of mesenchymal cell development and remodeling of the distal lung septae and arteries. Mutant lung histology at the saccular phase shows mesenchymal and saccular wall thickening, and abnormal proliferation of α-smooth muscle actin-positive cells, as well as increased mesenchymal/fibroblast cell differentiation, which become more sever when deleting both genes. Our study indicates that SHH but not TGF-β signaling pathway is a central mediator for the histologic alterations described in the saccular phenotype of Eya1−/− or Six1−/− lungs. Indeed, genetic reduction of SHH activity in vivo or inhibition of its activity in vitro substantially rescues lung mesenchymal and alveolar phenotype of mutant mice at the saccular phase. These findings uncover novel functions for Six1–Eya1–SHH pathway during the saccular phase of lung morphogenesis, providing a conceptual framework for future mechanistic and translational studies in this area.

Disturbance of post‐natal lung development in Norwich terriers

An arrest in the post‐natal lung development is a major cause of death in Norwich terrier puppies. Pedigree analysis indicates that the disorder is inherited. In dogs the lungs at term are in the saccular phase and the development into the alveolar stage occurs during the neonatal period.In the affected lungs the alveolar septae are poorly developed and the alveolar walls are thicker than normal. Immunohistochemical staining for vimentin, smooth muscle actin (SMA), CD31 and thrombomodulin was used to characterize the cells forming the alveolar parenchyme. There is a sequential and spatial pattern of SMA staining in normal canine lung examined 3 days, 3 weeks and 3 months after birth. The pattern appears to be similar in the affected puppies up to 3 weeks of age.Our canine model enables us to study a spontaneously occurring disease. The aim is to describe the genetic background of this congenital pulmonary disorder and to learn more about the molecular events involved in post‐natal lung development. In addition we wish to develop a DNA test for breeding purposes to eradicate the disease in Norwich terrier breed.

Sex-specific perinatal expression of glutathione peroxidases during mouse lung development

Reports indicate that antioxidant enzymes like the glutathione peroxidases (GPx) can be regulated by sex steroids. The GPx, a major class of antioxidants involved in H2O2 and lipid hydroperoxides neutralization, showed an age- and sex-specific expression in many adult organs including the lung. High levels of androgens in the male lung are known to delay the surge of surfactant synthesis during gestation in several species. However, the impact of male androgens on antioxidant GPx early in life remains to be determined. The objective was to study the lung sex-specific expression of GPx during BALB/c mouse perinatal development. The mRNA expression of four seleno-dependent Gpx (Gpx1 to 4) in the lung of both sexes was characterized by real-time PCR from gestational day 15 to postnatal day 30, covering the entire canalicular, saccular and alveolar stages. Immunohistochemistry of GPx-1, -3 and -4, and seleno-dependent GPx enzymatic assays were also performed in the lung. We found a transient lower Gpx1 mRNA level in male than in female lungs during the first 5 days after birth, corresponding to the saccular phase. This dimorphic expression was concomitant to a sex difference in GPx enzymatic activity corrected for blood. It is, to our knowledge, the first report of a sex dimorphism for murine lung enzymatic antioxidant defenses during the perinatal period.

Evolution and development of gas exchange structures in Mammalia: The placenta and the lung

Appropriate oxygen supply is crucial for organisms. Here we examine the evolution of structures associated with the delivery of oxygen in the pre- and postnatal phases in mammals. There is an enormous structural and functional variability in the placenta that has facilitated the evolution of specialized reproductive strategies, such as precociality. In particular the cell layers separating fetal and maternal blood differ markedly: a non-invasive epitheliochorial placenta, which increases the diffusion distance, represents a derived state in ungulates. Rodents and their relatives have an invasive haemochorial placental type as optimum for the diffusion distance. In contrast, lung development is highly conserved and differences in the lungs of neonates can be explained by different developmental rates. Monotremes and marsupials have altricial stages with lungs at the early saccular phase, whereas newborn eutherians have lungs at the late saccular or alveolar phase. In conclusion, the evolution of exchange structures in the pre- and postnatal periods does not follow similar principles.

Pulmonary Maturation in Canine Foetuses From Early Pregnancy to Parturition

Development of the foetal respiratory system includes both pulmonary growth and maturation. In human medicine, a higher incidence of respiratory distress is reported in newborn males. This study aimed to identify different phases of canine foetal lung maturation throughout pregnancy, to determine the stage of pregnancy in which surfactant production begins and to compare pulmonary development of male and female foetuses. Pregnant bitches (34) were subjected to elective ovariohysterectomy and allocated into four groups, according to the stage of pregnancy: 30-40 days of pregnancy (n = 10), 41-50 days (n = 10), 51-60 days (n = 10) and bitches in the first stage of parturition (n = 4). Foetal lungs were histologically processed and evaluated by optical microscopy. The pseudoglandular phase was identified between the 35th day and 46th day of gestation; the onset of canalicular and saccular periods was observed, respectively, from the 48th day and 60th day of pregnancy. Lungs from foetuses at term were in the saccular phase; thus, the development into the alveolar period occurs in the neonatal period. The histological analyses revealed that respiratory tract development is centrifugal, from upper to lower airways. Therefore, it is possible to identify distinct development periods in different portions of the same organ. In conclusion, the saccular phase of lung development begins around 57 and 60 days of pregnancy, the period in which surfactant production is believed to occur. Male and female foetuses present similar pulmonary development from early pregnancy until parturition.