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RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo.

Trichloroethylene (TCE) has previously been shown, in adult mouse lung, to alter the distribution of phospholipid associated with the pulmonary surfactant and to affect the activity of phospholipase A2, an enzyme involved in synthesis of the surfactant. However there are no data available on the effects of maternal exposure to TCE on fetal lung development. To determine if maternal TCE exposure may affect fetal pulmonary development, pregnant mice were treated with TCE (3000 mg/kg body wt.) administered intraperitoneally on day 17 of pregnancy. Fetuses were examined on the 18th and 19th gestational days and the 1st, 5th and 10th postnatal days. TCE caused increased mortality among 18- and 19-day-old fetuses and 1-day-old newborn mice. In addition mean body weight was reduced on the 1st postnatal day in animals exposed to TCE. Specific lung weights were also significantly reduced on both the 18th and 19th gestational days. More importantly, total lung phospholipid content was depressed immediately before birth; total DNA was not affected. These results suggest that prenatal maternal exposure to TCE may delay pulmonary maturation during the critical period when surfactant synthesis is initiated.

Fetal lung fibroblasts interact with type II epithelial cells, inducing their maturation. This interaction arises by secretion of factors which alter fetal type II cell function. To analyze these factors, conditioned medium (CM) was produced by exposing serum-free minimum essential medium, with [35S]methionine (5 microCi/ml), to confluent cultures of fetal rabbit lung fibroblasts. This medium was tested for ability to stimulate [3H]choline incorporation by fetal type II cells and subsequently fractionated on molecular weight filtration columns P60 (2.5cm x 90cm; NMW cutoff, 60kd; 1M acetic acid) and A1.5m (2.5cm x 90cm; NMW cutoff, 1,500kd; Tris-buffered saline) and a hydroxyapatite column (HT) (1.5cm x 30cm; NaCl and 0.01-0.3M phosphate). Crude medium stimulated choline incorporation into phosphatidylcholine. [35S]methionine was resolved in void volume material and in material of apparent molecular weight of 6000 daltons on the P60 filtration column. Filtration on the A1.5m column showed two major fractions with radiolabel incorporation. Each of these was resolved into two subfractions on HT chromatography. The high molecular mass fraction contained material which stimulated [3H]choline incorporation by fetal type II cells. The low molecular mass fraction tended to inhibit [3H]choline incorporation. The second subfractions of both the first and second primary fractions inhibited [3H]thymidine incorporation into DNA by fetal type II cells. SDS-PAGE electrophoresis and autoradiography showed that under reducing conditions, each peak contained several proteins. However few of these displayed radioactivity. These results indicate that protein factors produced by fetal lung fibroblasts may be involved in regulating both differentiation and replication of fetal type II cells.

beta-Aminopropionitrile (beta APN) was administered intraperitoneally to rats on postnatal days 1, 3, and 5. Body weight, lung volume, lung weight, number of alveoli per unit area and volume, total number of alveoli in the lung, and the total length of elastic fibers in the lung decreased, and the average alveolar volume increased in comparison with control animals similarly treated with saline. From Day 2 of age to Day 6 the total length of elastic fibers increased in control lungs but remained almost the same following beta APN treatment. Ultrastructurally, both the quality and quantity of elastin in lung alveolar wall were affected. beta APN also inhibited the synthesis of deoxyribonucleic acid in interstitial, endothelial, and Type II epithelial cells of the lung alveolar wall. The diminished number of alveoli gives support to the hypothesis that the elastin-collagen network may play a key role in postnatal alveolar multiplication.

The effect of starvation on postnatal lung growth in rats was investigated. Litters were starved twice, each time for 24 hours, on Day 1 and Day 5 after birth. One group of littermates was sacrificed on Day 7, and another group, on Day 14 of postnatal life. Intermittent starvation diminished lung growth. This was accompanied by reduced somatic growth. On postnatal Day 7, lung volume, total number of alveoli, and internal surface area of the lung were decreased in starved rats, but structurally their lungs appeared similar to control lungs. On postnatal Day 14, a striking morphologic difference was observed between the lungs of control and starved pups. Following starvation, retardation of lung growth was manifested in all the parameters studied. One week of normal uninterrupted suckling could not overcome the starvation-induced initial effects on growing lungs. It is concluded that starvation, soon after birth, affects adversely the normal development of lung.