Some observations on dermis development in fetal porcine skin

The study describes dermis development in fetal porcine skin between 40 d GA and birth. The first fibroblast arrangement occurs about 47 d GA, while between 60-65 d GA the first fibres are visible. They can be identified as collagen about 67 d GA, somewhat later the dermis shows 2 strata. At the end of median gestional life, the first birefringent lattice pattern of collagen fibre bundles is formed. The first elastic fibres cannot be demonstrated before 100 d GA.A clear increase in fibrocyte numbers occurs about 70-80 d GA, the next one at birth. The thickness development of the fetal porcine dermis is closely correlated with the increase in body weight. Additionally RNA and glycogen content developments have been studied in the fetal dermis.     

Structural and histochemical aspects of epidermis development of fetal porcine skin

Epidermal development of fetal porcine skin was studied in fetuses from 41 days of gestation until birth with scanning and electron microscopy techniques as well as histochemical methods, including immunohistochemistry. The porcine fetus develops a relatively thick and solid multilayered cover of epidermal cells, which is not lost before birth. It consists of tightly packed cells of the periderm and the stratum intermedium. The periderm cells are totally filled with filamentous proteins; in the intermediate cells, the filamentous proteins are concentrated in the cell periphery, forming a thick marginal zone. Immunohistochemically, the cytofilaments could be identified as cytokeratins of lower and higher molecular weights. The first thin stratum corneum lamellae are formed below the stratum intermedium at about 80–85 days of gestation.     

Morphometric analysis of fetal rat lung development

Applying the zone concept described previously, we quantitatively analyzed fetal rat lung development. The zone concept allowed us to coherently define reference spaces in the developing lung, a prerequisite for morphometric analysis. The peripheral zone I corresponds to a zone of growth of yet undifferentiated tissues; zone II arises from zone I and represents a region of structural and cellular differentiation; zones III and IV comprise the conducting airways and vessels. Lungs of fetal rats aged 17–23 days and 20 hours postnatal were fixed with OsO₄ and glutaraldehyde and processed for light and electron microscopic morphometry implemented by point and intersection counting. Fetal lung volume grew in proportion to body weight. Zone II being the largest compartment, its volume changes largely determined lung growth rates. Zone II increased in mass owing to differentiation processes at the interface to zone I where the proximal portions of zone I were continuously shifted into zone II by differentiation. New tissue was generated within zone I. Due to these combined processes zone I changed little in volume until it disappeared at the end of the canalicular stage. The presence in the pseudoglandular stage of half of the parenchymal epithelial mass available around birth indicated that parenchymal development started earlier than assumed so far. While the endothelial surface increased most at birth, the epithelial surface grew by more than 600% at day 21, reflecting the onset of canalisation. The study confirmed the usefulness of the zone concept for morphometry and provided some new insights into lung development. © 1993 Wiley-Liss, Inc.     

Morphometry of postnatal development in the porcine lung

Alveolar regions of normal pig lungs (newborn to 60-day-old) were characterized morphometrically to provide a basis for comparison in future investigations of porcine respiratory diseases. Endotracheal installation of fixative was done to expand the lungs uniformly at total capacity. Differential effects of lobar variations were determined by stratified random sampling of lung lobes. A stereologic study was done by point and intersection counts on electron micrographs. At birth, the lungs were remarkably well developed. Relative alveolar and capillary surface densities and air-blood tissue barrier thicknesses were at adult levels. In allometric regressions, volumes and surfaces of lung components regressed directly to lung volume, but monoexponentially (to the 3/4 power) with body weight. In the first postnatal week, however, relative volume densities of cellular interstitium in septal tissue and of capillary lumina in parenchyma increased at statistically significant levels. Composition of lung parenchyma and septa was changed, although without statistically significant direct impact on parameters related to gas exchange. Type II pneumocytes had increased nuclear to cytoplasmic volume ratios in 7- to 14-day-old pigs, probably reflecting cell activation and increased surfactant production. Age (postnatal lung growth) created the most substantial variance of results; interanimal variation in pigs of the same age was less important and no consistent lobar variations were seen.     

Structural analysis of fetal rat lung development.

The primary aim of this morphological investigation was to elaborate a concept allowing us to coherently define reference spaces for morphometric analysis of fetal lung development. Beyond this quantitative goal, morphological analysis of cell types, definition of compartments, and reflection about the prospective fate of their constituents provided per se new insights into the developmental processes. Lungs of rat fetuses aged 17-23 days and newborns aged 20 hours were fixed with an osmium tetroxide and glutaraldehyde mixture and their volume determined. Left lungs were embedded in Epon and investigated by light and electron microscopy. The right lung of one animal per group was embedded in methacrylate and step sections obtained to precisely locate the airways within the mesenchyme. The various cell types, their topographical relationships, and their morphological alterations with ongoing development were analyzed with regard to their prospective potentials of differentiation. The developing lung could be partitioned into four zones further subdivided into defined compartments. Zone I forms a superficial mantle around the lobes and the future acini. Consisting of primitive mesenchymal cells, it represents a zone of growth which disappears with the onset of the saccular stage. Zone II is mainly a zone of differentiation. Its interstitium stains intensely due to a dense population of dark cells. Up to gestational day 19, zone II contains future conductive airways with their vessels. After day 21, it comprises the whole prospective gas exchange region. Zones III and IV contain the elements of the airway tree and vascular system, zone IV corresponding to the most proximal generations with an adventitial layer. For all differentiation processes, a centrifugal directionality is manifested.     

Glucocorticoids and hypothyroidism modulate development of fetal lung insulin receptors

We investigated the development of insulin receptors in membranes of fetal rabbit lung during normal ontogeny and the effect of glucocorticoids and hypothyroidism. Specific binding of 125I-insulin to fetal lung membranes increased progressively to a peak at 29 days gestation, declining by 30 days. Scatchard plots were curvilinear and revealed a progressive increase in receptor numbers (X 10(10)/mg protein) from 129 +/- 7 (mean +/- SE) at 22-24 days to 575 +/- 16 at 29 days, declining to 467 +/- 12 at 30 days, term being approximately 31 days. Affinities did not change throughout gestation and were similar to those of adult lung; receptor numbers in adults were significantly lower than in fetuses at 26-30 days. Epinephrine and PGE1 could evoke a doubling of cAMP production in adult and fetal lung membranes until 29 days. Concomitantly with the fall in fetal insulin receptor number at 30 days, cAMP production in response to epinephrine or PGE1 increased fivefold. Induction of fetal hypothyroidism decreased insulin receptor numbers in the lung of the 28-day fetus by 70% from control (P less than 0.001) without a change in receptor affinity. In contrast, betamethasone administration increased fetal lung insulin receptor numbers by 250% (P less than 0.001) but did not alter their affinity; maternal lung insulin receptors were not altered. Thus, normal ontogeny of the fetal lung insulin receptor is characterized by a progressive increase in number followed by decline immediately before parturition associated with a sharp increase of cAMP responsiveness of the membranes. Hypothyroidism and glucocorticoid exposure can modulate the normal development of the fetal lung insulin receptor.     

水通道蛋白5在人胎肺发育中的表达及意义

目的检测水通道蛋白5（AQP5）在胎儿正常肺组织和发育异常肺组织的表达,探讨AQP5对胎肺发育的影响及其在肺液代谢中的作用。方法应用RT-PCR检测AQP5mRNA在各胎肺中的表达以及real-timePCR检测AQP5在正常胎肺和发育异常胎肺中的基因表达差异。结果AQP5mRNA在各孕周胎肺均有表达,发育异常胎肺组织较正常胎肺组织表达量明显减少,差异有统计学意义（P〈0.05））。结论发育异常胎肺AQP5表达下降,其对胎肺的正常发育和肺液代谢可能有重要影响。     

A morphologic and morphometric analysis of fetal lung development in the sheep

In the sheep, fetal lung development proceeds to a later stage of maturity than in smaller laboratory animals. Of the four stages in pulmonary development recognizable in this species–embryologic, pseudoglandular, canalicular, and aveolar–the latter three are described in the present study using histologic, morphometric, and ultrastructural techniques. During the pseudoglandular stage, the major airways developed centrifugally. Cartilaginous, glandular, muscular, vascular, and neural elements were present in major airway walls from an early age. During the canalicular stage, volume expansion of the lung was accomplished by rapid growth of large terminal spaces. In the final stage, alveoli were formed following subdivision of the large terminal spaces by alveolar crests. The alveolar lining epithelium differentiated during the latter two stages producing a large increase in alveolar surface area, particularly during the alveolar stage; a large increase in pulmonary capillary surface area also accompanied alveolar development. Thus, just prior to birth, the fetal sheep lung has a well-developed airway system and alveolar network, in preparation for postnatal gas exchange.     

Embryonic essential myosin light chain regulates fetal lung development in rats

Congenital diaphragmatic hernia (CDH) is currently the most life-threatening congenital anomaly the major finding of which is lung hypoplasia. Lung hypoplasia pathophysiology involves early developmental molecular insult in branching morphogenesis and a late mechanical insult by abdominal herniation in maturation and differentiation processes. Since early determinants of lung hypoplasia might appear as promising targets for prenatal therapy, proteomics analysis of normal and nitrofen-induced hypoplastic lungs was performed at 17.5 days after conception. The major differentially expressed protein was identified by mass spectrometry as myosin light chain 1a (MLC1a). Embryonic essential MLC1a and regulatory myosin light chain 2 (MLC2) were characterized throughout normal and abnormal lung development by immunohistochemistry and Western blot. Disruption of MLC1a expression was assessed in normal lung explant cultures by antisense oligodeoxynucleotides. Since early stages of normal lung development, MLC1a was expressed in vascular smooth muscle (VSM) cells of pulmonary artery, and MLC2 was present in parabronchial smooth muscle and VSM cells of pulmonary vessels. In addition, early smooth muscle differentiation delay was observed by immunohistochemistry of alpha-smooth muscle actin and transforming growth factor-beta1. Disruption of MLC1a expression during normal pulmonary development led to significant growth and branching impairment, suggesting a role in branching morphogenesis. Both MLC1a and MLC2 were absent from hypoplastic fetal lungs during pseudoglandular stage of lung development, whereas their expression partially recovered by prenatal treatment with vitamin A. Thus, a deficiency in contractile proteins MLC1a and MLC2 might have a role among the early molecular determinants of lung hypoplasia in the rat model of nitrofen-induced CDH.     

Epithelial-interstitial cell interactions in fetal rat lung development accelerated by steroids

Direct cell to cell contact has been suggested as the means whereby fibroblast factors influence epithelial cell differentiation in the developing lung. To obtain further evidence for this hypothesis, the role of epithelial-mesenchymal cell contacts is now examined when lung development is accelerated after steroid injection. Male and female rat fetuses were studied from day 17 to day 22 of gestation. The fetuses were from mothers injected with 2 mg/kg of dexamethasone at 2 days before death and [3H]thymidine at 1 hour before death. Steroid injected during the rapid growth phase reduced total DNA in fetal lung, and from autoradiographs, the effect was most noticeable in epithelial cells. Differentiation began sooner after steroid; more epithelial cells contained lamellar bodies and disaturated phosphatidylcholine levels were higher. From quantitative ultrastructural studies, these changes in phospholipid correlated with an increased frequency of epithelial-interstitial cell contacts. Steroid treatment did not abolish the sex-related difference seen in controls; both males and females showed increases in cell to cell contacts and in lipid synthesis in the lung. The results demonstrate that acceleration of epithelial cell maturation with increased surfactant synthesis is associated with increased epithelial-interstitial cell contacts. This supports the concept that regulatory messages from the fetal fibroblast are passed directly to specific epithelial cells to initiate surfactant synthesis.     

Expression profile of microRNAs in fetal lung development of Sprague-Dawley rats

As well-known regulators of gene expression, microRNAs (miRNAs) play an important role not only in cell proliferation and differentiation, but also in tumorigenesis and organ development. Furthermore, it is estimated that miRNAs may be responsible for regulating the expression of nearly one-third of the human genome. Simultaneously, in the clinic, with advances in neonatal care, a larger number of premature infants are being saved, and thus diseases of lung development, including bronchopulmonary dysplasia (BPD) have become more and more common. However, only a few miRNA studies have studied their connection with diseases of lung development. In our study, we used a miRNA microarray including more than 1891 capture probes to profile the expression of miRNAs at three time points of rat lung development [embryonic (E) Day 16 (E16), E19, E21]. miRNAs found to have consistent fold-changes (fold-change>2.0) during all three time points were selected and validated by real-time PCR. As a result, 167 differentially expressed miRNAs were found during rat lung organogenesis, including 81 upregulated and 86 downregulated miRNAs. Seven miRNAs were selected and characterized by having a consistent >2-fold changes between all three groups. Among these 7 miRNAs, except for let-7a, the other 6 miRNAs (miR-1949, miR-125b-5p, miR-296, miR-93, miR-146b, miR-3560) are all first reported for the first time in lung development. Finally, due to the fact that they demonstrated higher fold changes, from these 7 miRNAs we selected miR-125b-5p, miR-296, miR-93, miR-146b and miR-3560 for real-time PCR. We hypothesized that these newly identified miRNAs may play an important role in fetal lung development, and this experimental result could help us to further clarify the mechanism of normal lung development including the development of type?II pneumocytes. This may provide a physiological basis for future research on diseases of lung development.     

Cytopathogenicity of Mycoplasma hyopneumoniae in porcine tracheal ring and lung explant organ cultures alone and in combination with monolayer cultures of fetal lung fibroblasts.

Porcine tracheal rings and lung explants alone and in combination with monolayer cultures of porcine lung fibroblasts (PLF) were separately inoculated with virulent strains of Mycoplasma hyopneumoniae and incubated at various times. The preparations were observed by bright-field, phase-contrast, and scanning electron microscopy. In PLF cultures, the strains at initial concentrations of 10(1.3) colony-forming units/ml increased within 3 days to 10(6) colony-forming units/ml, showed progressive clustering on the cells, and caused some sloughing. Introduction of a tracheal ring or lung explant into these mycoplasma-infected PLF cultures caused the explant to lose its epithelial ciliary motility. Eventually parts or whole cells of the respective ciliated epithelium were lost. Without infected PLF monolayers, the explants inoculated with M. hyopneumoniae were less susceptible to infection. When uninfected explants were incubated for 18 days or kept in stock for 2 months, they did not show the above changes. With 5 h postinoculation, M. hyopneumoniae cultures became intimately associated with the PLF culture, but when epithelial cell sloughing occurred, the mycoplasmal cells became dependent on the introduction of a fresh PLF monolayer or a tracheal or lung explant for survival.     

Toll-like receptor signaling inhibits structural development of the distal fetal mouse lung.

We tested the hypothesis that innate immune signaling in utero could disrupt the structural development of the fetal lung, contributing to the pathogenesis of bronchopulmonary dysplasia. Injection of Escherichia coli lipopolysaccharide (LPS) into the amniotic fluid of E15 BALB/cJ mice increased the luminal volume density of fetal mouse lungs at embryonic day (E) 17 and E18. LPS also increased luminal volume and decreased distal lung branching in fetal mouse lung explants. This effect required NF-kappaB activation and functional Toll-Like Receptor 4. Airway branching may require fibronectin-dependent epithelial-mesenchymal interactions, representing a potential target for innate immune signaling. Anti-fibronectin antibodies and LPS both blocked distal lung branching. By immunofluorescence, fibronectin localized to the clefts between newly formed airways but was restricted to peripheral mesenchymal cells in LPS-exposed explants. These data suggest that LPS may alter the expression pattern of mesenchymal fibronectin, potentially disrupting epithelial-mesenchymal interactions and inhibiting distal airway branching and alveolarization. This mechanism may link innate immune signaling with defects in structural development of the fetal lung.     

The growth factor midkine is modulated by both glucocorticoid and retinoid in fetal lung development

The glucocorticoids (GC) and retinoids (RA) modulate branching morphogenesis and cytodifferentiation in the developing lung. We investigated downstream target genes that link glucocorticoid stimulation to the achievement of a mature lung in glucocorticoid receptor (GR) knockout mice. All GR(null) mice and approximately 80% of mice homozygous for a hypomorphic allele (GR(hypo)) die shortly after birth of respiratory failure. cDNA microarray analysis showed organ-specific upregulation of the retinoic acid responsive gene midkine (MK) and its chondroitin-sulfate binding partner PG-M/versican at fetal day 18 and at neonatal day 1 in lungs of GR(hypo) mice, and at neonatal day 1 in lungs of GR(null) mice. By contrast, lung MK and PG-M/versican were downregulated in these mice at fetal day 16.5. In situ hybridization studies showed a dramatic decrease in MK and PG-M/versican RNA between days 16.5 and 17.5 in GR(WT) but not in GR(null) mice. Continued diffuse and robust expression of MK protein was observed in GR(null) mice at neonatal day 1. These findings suggest that MK may contribute to the dysmature lung phenotype in GR-deficient mice. Exposure of cultured day 21 fetal rat lung cells to GC downregulated MK, whereas RA enhanced MK expression. Our findings demonstrate the coincident modulation of expression of MK at the same developmental time point by both GC and RA, providing a potential mechanism for the integration of GC and RA effects on fetal lung development.     

The neonatal porcine lung: ultrastructural morphology and postnatal development of the terminal airways and alveolar region

Morphology and postnatal development of the porcine lung are described in animals ranging in age from newborn through 60 days. Standardized fixation was accomplished by intratracheal instillation of glutaraldehyde under constant pressure. Light microscopic, scanning, and transmission electron microscopic investigations revealed that the porcine lung follows the common architecture of mammalian lungs, but has certain peculiarities as well: intravascular macrophages, ultrastructurally similar to Kupffer cells, are attached to endothelial cells in pulmonary capillaries and are involved in erythrophagocytosis during the first postnatal weeks. Type II pneumocytes of newborn pigs exhibit signs of cell activation, mainly complex nuclear bodies in the cell nuclei. At the same time high levels of 17-hydroxycorticosteroids are observed in the newborn blood plasma. Terminal airways of the porcine lung are nonalveolarized and are, therefore, of purely conductive function. At birth the porcine lung exhibits a high degree of maturity, and thick-walled primary saccules, as described in newborn rodents, are not seen. Septa appear straight and smooth, owing to rare ramification. Septal buds are discernible, and two capillary networks visible on both sides of septal cross sections are seen. Further subdivision of the airspaces occurs in the first two postnatal weeks. Precociousness and fast postnatal growth of the porcine species are assumed to be the reason of this advanced degree of lung maturity at birth and the following rapid pulmonary development.     

Distribution of ERK1/2 and ERK3 during normal rat fetal lung development

The extracellular regulated kinases-1 and -2 (ERK1/2) are well-characterized mitogen-activated protein kinases (MAPK) that play critical roles in proliferation and differentiation, whereas the function(s) of MAPK ERK3 are currently unknown. To understand better the roles of these kinases in development, the temporal distribution of ERK1, -2, and -3 proteins were investigated in multiple tissues. The ERK3 protein, in contrast to ERK1/2 varied both between and within individual organs over time. To characterize this variability in greater detail, the temporal and spatial distributions of activated ERK1/2 and ERK3 during rat fetal lung development were investigated. The diphosphorylated (activated) forms of ERK1/2 (dp-ERK1/2), ERK3, and its phosphorylated form (P-ERK3) decreased from embryonic day 17 (E17) through E21 while both ERK1 and ERK2 total proteins remained unchanged, indicating that ERK1/2 and ERK3 proteins are expressed independently during fetal lung development. In addition, characterization of the distribution of these proteins by fluorescent immunohistochemistry indicated that phosphorylated ERK1/2 and total ERK1/2 were distributed throughout multiple cell types, with the phosphorylated ERK1/2 colocalizing with prophase mitotic cells. In contrast, ERK3 was restricted to the distal lung epithelium during the pseudoglandular phase (E17) but shifted to the proximal airways, particularly Clara cells during the saccular stage (E21). The P-ERK3 colocalized with the mitotic marker P-histone H3 in fetal lung and in NIH3T3 and HeLa cells, implicating a potential role for P-ERK3 in mitosis. Thus, expression of ERK1/2 and ERK3 and their phosphorylated forms are expressed independently and are temporally and spatially localized during fetal lung morphogenesis. These observations will facilitate detailed functional analysis of these kinases to assess their roles in pulmonary development and diseases.     

The development of alveolar septa in fetal sheep lung. An ultrastructural and immunohistochemical study

The morphogenesis of pulmonary alveolar septa in the sheep was studied by light microscopy, transmission electron microscopy and light microscopic immunohistochemistry for the detection of elastin. The primordia of alveolar septa developed in the glandular stage in areas subjacent to the epithelium, and formed alveolar septa by protruding into the glandular lumina. In their earliest stage, the primordia consisted of groups of fibroblasts, which were associated with elastic fibers and unit collagen fibrils and were surrounded by epithelial basement membrane and by more immature fibroblasts. The fibroblasts in the primordia subsequently became myofibroblasts or smooth muscle cells. In the alveolar zone of the glands, elastic fibers were exclusively found in the primordia of alveolar septa in early developing lung. In early developing lung, wavy, thickened epithelial basement membranes were found in the regions of the glands, which eventually underwent considerable expansion of their surface areas, especially in the primordia of alveolar septa and the bifurcations in the alveolar zones. Areas of fusion of the basement membranes of capillary endothelial cells and epithelial cells in the alveolar zone were found after the formation of the primordia of alveolar septa was accomplished. These areas of fusion were not found in the primordia themselves, but in regions between the primordia. Epithelial cell flattening and differentiation occurred after the formation of the primordia of alveolar septa, and flattening was first observed in the areas of the primordia and the bifurcations of the alveolar zones.