Cystic Fibrosis Gene and Protein Expression during Fetal Lung Development

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) transports Cl- in the apical membrane of secretory epithelial cells. Normal lung development is critically dependent on active Cl- secretion by developing airway epithelia. In this study, the polymerase chain reaction (PCR) was used to detect CFTR mRNA expression in rabbit lung at different stages of development. Using nested amplification in exons 7 and 11, a 494-bp fragment was isolated from cDNA prepared from lungs obtained at gestational days 22 (pseudoglandular) and 29 (terminal sac). Nucleotide sequencing verified the identification of rabbit CFTR sequences. At day 22 of gestation, CFTR was detected in fetal liver, kidney, heart, intestine, and brain. CFTR could not be amplified from adult rabbit brain. CFTR protein was detected using a polyclonal antibody raised in chickens against a synthetic peptide from the R domain conjugated to thyroglobulin. Rabbit CFTR migrated as a 165-kD protein on 5% sodium dodecyl sulfate polyacrylamide gels when detected either by chicken anti-R IgG or a previously characterized rabbit anti-R antibody. In frozen sections of fetal rabbit lung at day 22 of gestation, CFTR was localized to the membrane regions of primitive tubular epithelial cells and absent from surrounding connective tissue. At day 29, CFTR was concentrated in the apical region of bronchiolar epithelial cells and absent from alveoli and vessels. These results suggest that CFTR gene expression begins very early in lung development and, with differentiation of the airways, becomes confined to differentiated bronchiolar epithelium, the presumed site for cAMP-mediated Cl- secretion.

Developmental Differences of Cystic Fibrosis Transmembrane Conductance Regulator Functional Expression in Isolated Rat Fetal Distal Airway Epithelial Cells

“…Volume reduction activated by Ca2+ ionophore was not detectable at d 18 and 19, but could be observed in cells at d 20 and 2 1 suggesting that the Ca2+-activated C1-conductance is developmentally expressed as these epithelial cells differentiate from a net secretory to absorptive phenotype (4). In rabbit fetal lung at gestational age d 22 (pseudoglandular) CFTR was localized to the membrane regions of tubular epithelial cells but at d 29 (terminal sac) was concentrated in the apical region of bronchiolar epithelial cells and absent from alveoli (32). These results are consistent with our findings in rat fetal lung.…”

CFTR gene transduction in neonatal rabbits using anadeno-associated virus (AAV) vector

“…These antisera have been previously characterized in our laboratory. 3,11 Antitrates with scattered neutrophils, eosinophils and mononuclear cells. Figure 5b and c are representative of n = 4 serum 169 will specifically immunoprecipitate human CFTR from transfected heterologous cells (Cheng SE, control animals, and Figure 5d is representative of n = 8 vector-treated animals (dose range 10 9 -10 10 particles).…”

Immunohistochemical Localization of Cystic Fibrosis Transmembrane Conductance Regulator in Human Fetal Airway and Digestive Mucosa

“…CFTR could be implicated in none- Oh, pithelial cells such as muscle or enteric nervous plexus in the intestinal wall. Recently, CFTR transcripts were detected in a variety of secretory and nonsecretory organs, such as brain and heart, in fetal rabbits (20). The localization of CFTR and the intensity of the immunoreactivity in the airways were different throughout the successive periods of development and differed in the various parts of the airways.…”