Human Molecular Genetics, 2002, Vol. 11, No. 2 125-131
© 2002 Oxford University Press
Temporal regulation of CFTR expression during ovine lung development: implications for CF gene therapy
Paediatric Molecular Genetics, Institute of Molecular Medicine, Oxford University, John Radcliffe Hospital, Oxford OX3 9DS, UK, 1Nuffield Department of Clinical Laboratory Sciences, Oxford University, John Radcliffe Hospital, Oxford OX3 9DU, UK and 2Growth and Development Unit, Field Laboratory, Oxford University, Oxford OX2 8QJ, UK
Received September 10, 2001; Revised and Accepted November 13, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a small conductance chloride ion channel that may interact directly with other channels including the epithelial sodium channel (ENaC). CFTR is known to be more abundant in the airway epithelium during the second trimester of human development than after birth. This could be a consequence of the change in function of the respiratory epithelium from chloride secretion to sodium absorption near term. Alternatively it might reflect an additional role for CFTR in the developing airway epithelium. Though the lung epithelia of CF fetuses and infants rarely show gross histological abnormalities, there is often evidence of inflammation. Our aim was to establish whether CFTR expression levels correlated with specific developmental stages or differentiated functions in the ovine fetal lung. We evaluated CFTR expression using a quantitative assay of mRNA at 14 time points through gestation and showed highest levels at the start of the second trimester followed by a gradual decline through to term. In contrast, ENaC expression increased from the start of the third trimester. These results support a role for CFTR in differentiation of the respiratory epithelium and suggest that its expression levels are not merely reflecting major changes in the sodium/chloride bulk flow close to term. These observations may have significant implications for the likely success of CF gene therapy in the postnatal lung.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a small conductance cAMP-activated chloride ion channel (1). CFTR protein is located primarily in the apical membranes of epithelial cells within the respiratory and digestive systems. About 1000 disease-associated mutations have been defined in the CFTR gene (www.genetics.sickkids.on.ca) and the majority are associated with characteristic airway disease, pancreatic insufficiency, male infertility and elevated levels of sodium chloride in sweat. It is difficult to explain how mutations in a single small-conductance epithelial chloride channel cause the pleiotropic manifestations of this disease, though a complex picture is now emerging in which CFTR interacts directly or indirectly with additional proteins. CFTR is known to regulate other ion channels including the epithelial sodium channel (ENaC) (2) and the volume-activated chloride channel (3). CFTR may also be involved in other cellular processes, for example, membrane recycling (4,5) and apoptosis (6,7).
Relatively little attention has been given to progression of CF disease in utero though this affects postnatal pathology and may be of relevance to the likely success of gene therapy. The initial pathology of CF is exhibited in the pancreas in the early second trimester of gestation, when proteinaceous material starts to obstruct the ductal tree (8,9). This is perhaps not surprising since CFTR is expressed in epithelia from before the start of the second trimester of human development and CFTR expression levels are high in the mid-trimester pancreas (10–12). In most tissues the CFTR expression levels show little fluctuation between the second trimester of gestation and postnatal life. However, CFTR appears to be developmentally regulated in both human and sheep lung epithelium (11–13) where high expression levels during fetal life are in contrast to much lower abundance of CFTR mRNA by birth, and into adulthood. The significance of this down-regulation of CFTR expression is the major focus of this work.
Since it is not feasible to investigate human lung development in utero, we have used the sheep to provide a useful model of human lung development (14). The sheep lung is anatomically more similar to human than that of the mouse, having submucosal glands throughout the cartilagenous airway. The expression pattern of CFTR in sheep is similar to humans, both temporally and spatially and we have shown that the ovine CFTR gene sequence shows close homology to the human gene (14,15). Expression of CFTR during human and sheep lung development has so far been assessed primarily by means of in situ hybridization (11,12), northern blotting (10,13) and reverse transcriptase–polymerase chain reaction (RT–PCR) (10,13). CFTR expression levels are high in the lung during the second trimester of gestation and low postnatally. To date, developmental expression of CFTR has only been examined at a limited number of time points and data have not been accurately quantitative. Our aim was to evaluate CFTR expression at specific time points throughout gestation using a real-time quantitative RT–PCR assay provided by TaqMan technology. We have generated a developmental profile of CFTR mRNA in sheep with particular attention to the down-regulation of CFTR in the respiratory system. This profile has identified key gestational ages where CFTR expression is maximal and hence when targeting of potential in utero therapies might be of most benefit.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Semi-quantitative RT–PCR of CFTR
In preliminary experiments CFTR expression during ovine gestation was evaluated by semi-quantitative RT–PCR. Primers C2R and C2L (16) that span exon 13 of the CFTR cDNA were used to reverse transcribe and amplify CFTR from RNA samples extracted from ovine tissues at 52, 66, 83, 94, 108, 129 and 143 days gestation, 147 (term) days and 2 days post-term. Subunit c of the ovine ATP synthase (17) was used as a reference standard for these RT–PCR reactions. The developmental profile of CFTR mRNA in lung tissue (Fig. 1) shows a decline between 83 days of gestation (mid-trimester) and term. Since semi-quantitative RT–PCR has limited power in providing accurate data on relative expression levels of a gene, we then used real-time (TaqMan) RT–PCR to quantify the precise developmental expression of ovine CFTR in the lung.
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Validation of the real-time CFTR RT–PCR assay target
The region of the ovine CFTR gene chosen for generating the TaqMan amplifying primers and probe covered the boundary between exons 6a and 6b. Since the CFTR gene is known to exhibit a range of alternatively spliced forms in a number of species (18) it was necessary to confirm that no aberrant transcripts lacking parts of exons 6a and 6b were produced from the ovine gene. Conventional RT–PCR was carried out using primers spanning exons 4–7 and no alternative transcripts lacking exons 6a or 6b were detected in RNA from lung at mid-gestation or birth, pancreas, small intestine, salivary gland and colon at 2 days after birth (data not shown). These results suggested that the real-time TaqMan RT–PCR assay targeted to the exon 6a/6b boundary, would accurately reflect total CFTR mRNA transcription.
Real-time RT–PCR for CFTR mRNA in ovine fetal tissues
The real-time RT–PCR assay for ovine CFTR mRNA was optimized and its reproducibility established. Values obtained from reactions in which template was omitted, consistently fell below the range of the standard curve and were <0.4% of experimental values. Standard curves were highly reproducible for the ovine CFTR and ribosomal RNA assays (R2 = 0.99) and genomic contribution to the RT–PCR reactions was <0.1%.
Two series of experiments were performed: the first evaluated CFTR expression in ovine lung tissues throughout the developmental time course; the second evaluated CFTR expression in eight ovine tissues at 83 and 108 days of gestation and at 2 days post-term.
Developmental expression of ovine CFTR in the lung
The developmental profile of CFTR expression in the lung showed a gradual fall in mRNA levels though the 147 days of gestation (Fig. 2). Values at 52 days of gestation, the end of the first trimester, were 75-fold higher than those at birth. By 66 days, CFTR mRNA levels were reduced 4.5-fold before increasing between 74 and 80 days. After levels peaked at 80 days of gestation, there was a gradual decline toward birth with negligible levels of expression by 4 days before term (143 days) and at birth. Two days after parturition, levels of CFTR mRNA increased slightly and this expression level was maintained at 2 weeks of age and in adult sheep tissue. Experiments were performed three times with samples analysed in triplicate. To evaluate the variation in CFTR expression levels in individuals at specific time points, samples were analysed from a sib pair at 74 days gestation and a half-sib pair at 2 days after birth. In both cases, CFTR expression levels were not significantly different from each other (74 days, P = 0.69; 2 days, P = 0.86; two-tailed Student’s t-test).
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Developmental expression of ovine CFTR in other organs
The developmental expression of ovine CFTR in pancreas, small intestine, colon, trachea, salivary gland, kidney and muscle was evaluated at three different time points (83 and 108 days of gestation and 2 days post-term) (Fig. 3). Muscle provided a negative control and as expected had negligible amounts of CFTR mRNA expression for all three time points in these experiments. Experiments were performed three times with samples analysed in triplicate.
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These are the first quantitative data that enable comparison of CFTR expression levels between tissues of the one species. They confirm the observation that the levels of CFTR mRNA in the pancreas and digestive system are significantly higher (at least 5-fold) than in the lung epithelium even at the time of maximal expression in the respiratory system. The observation of greater levels of CFTR mRNA in the colon than in the small intestine is probably a unique feature of the sheep and its digestive system as there are data to show the opposite distribution in the human intestine (11). Glandular organs such as pancreas, lung and salivary gland had a similar pattern of expression characterized by higher expression early in gestation followed by a decline towards birth with levels maintained 2 days post-term. CFTR mRNA levels were higher in both pancreas and salivary gland than in lung. In trachea CFTR mRNA levels were comparable to values in lung during gestation but rose at 2 days post-term. Small intestine expressed lower levels of CFTR mRNA before birth in comparison to 2 days after birth. Colon initially expressed very high amounts of CFTR mRNA, which then decreased slightly at mid-gestation before rising again to a high level after birth. CFTR mRNA expression in the ovine kidney is very low and declines between 83 and 2 days post-term.
Developmental expression of ovine ENaC in the lung
The CFTR and ENaC ion channels are thought to interact directly (2), hence it was pertinent to evaluate also the developmental expression of ENaC in the ovine lung. These assays were performed using conventional RT–PCR rather than real-time TaqMan RT–PCR due to lack of full sequence information for ovine ENaC and reported alternative splicing of this gene (19). In marked contrast to CFTR, expression of the 
subunit of ENaC is very low in the ovine lung at 52 days of gestation, but gradually rises through 108, 129, 143 and 147 (term) days of gestation to much higher levels in 2 days post-term and adult lung (Fig. 4). Again, subunit c of the ATP synthase was used as a reference standard (17) for these semi-quantitative RT–PCR reactions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have shown using semi-quantitative and quantitative RT–PCR that the high levels of CFTR expression seen in the second trimester ovine fetal lung decrease gradually through to term rather than declining suddenly near parturition. The detection of significant CFTR expression during lung development with low levels after birth confirms previous observations in humans and sheep (11–13). Tebbutt et al. (13) showed, by RT–PCR and northern blots, that CFTR mRNA expression in ovine fetal lung was far higher than after birth and into adulthood, and that the high levels seen at
70 days of ovine gestation were comparable to CFTR mRNA in mid-trimester human fetuses. However, the previous data did not evaluate CFTR expression throughout gestation. The gradual decline in CFTR that we observed during ovine fetal lung development could be accounted for by at least two explanations. Either, the percentage of cells that express CFTR within the lung epithelium decrease with time or the levels of expression within the same cell type gradually decreases. On the basis of previous data we would expect the former explanation to be correct. In postnatal human lung epithelium significant CFTR expression is limited to specific cells within the serous portion of submucosal glands and individual cells in the airway surface epithelium (20,21). This is in contrast to the apparent expression of CFTR mRNA in the majority of cells in the fetal lung epithelium (11). Furthermore, in human trachea, CFTR expression was seen to be intermediate and diffuse during the first trimester by in situ hybridization (11). In the second trimester, CFTR mRNA expression was reduced and limited to a subset of cells in the epithelia of the trachea and by the third trimester, only individual cells expressed CFTR but at very high levels (12).
High levels of CFTR mRNA expression have been observed by in situ hybridization in pancreas, small intestine and colon from an early gestational age in humans (11). These expression levels remain constant throughout gestation in contrast to lung expression patterns. The temporal and spatial expression of CFTR mRNA in ovine small intestine, pancreas, colon, salivary gland and kidney and muscle seen in our experiments, closely parallel the human gene (13). However, the quantitative assays show that in contrast to the human CFTR gene, which is expressed at a higher level in the crypts of the small intestine than in the colon, ovine CFTR mRNA is most abundant in the colonic epithelium. This may reflect functional differences in the ruminant digestive system. It is of interest that the decline of CFTR expression observed in ovine fetal lung between 83 days gestation and 2 days after birth is also seen in pancreas and salivary gland. CFTR may have a significant role in the development and differentiation of other glandular tissues.
The physiological implications of the gradual decline in lung CFTR expression levels during gestation warrant further discussion. The fetal lung is filled with a high chloride, low pH fluid that is distinct from amniotic fluid. Chloride is actively secreted from the lung epithelium into the lung lumen during development and as a chloride ion channel, CFTR protein could contribute to this process. However, chloride conductances other than CFTR are likely to be more important in fetal lung fluid secretion since CF is not associated with hypoplastic lungs at birth. Furthermore, the profile of CFTR expression during gestation does not correspond to changes in lung liquid secretion (22,23).
Postnatally, the major ion flows across the airway epithelium are uptake of sodium. Lung liquid begins to be reabsorbed by ovine lung epithelium at 140 days of gestation. Sodium reabsorption is achieved primarily by sodium channels located on the apical surface of the lung epithelium, the main one being the ENaC (24). Recent data have shown that ENaC is inhibited by CFTR, which probably interacts directly with it (2,25,26). The gradual decline in CFTR mRNA levels and protein would be compatible with enabling ENaC activity. Furthermore, we have shown that the abundance of ENaC transcripts is inversely proportional to the levels of CFTR mRNA in the developing ovine lung after the second trimester, hence the expression of the genes encoding these two ion channels may be also coordinately regulated.
An additional possibility is that CFTR expression in the fetal lung epithelium has an important, as yet undefined functional role in normal lung development. Though at birth lung pathology is not usually evident in CF, absence of functional CFTR protein in the developing lung has been reported to produce a pro-inflammatory state (27). Increased levels of interleukin-8 were observed in the airway surface fluid of human CF fetal tracheal grafts implanted into severe combined immunodeficient (SCID) mice. Though this apparent pro-inflammatory state of the epithelia did not result directly in histological abnormalities, challenge with Pseudomonas aeruginosa provoked an exaggerated immune response resulting in sloughing of epithelial cells and increased leukocyte activity, thereby increasing likelihood of further bacterial colonization (27).
The high levels of CFTR expression early in gestation may correspond to an important function of CFTR protein unique to this developmental stage. The periods of high and low CFTR expression that we have defined, correspond to distinct developmental phases in the sheep lung. Relatively high levels of CFTR mRNA expression are observed during the pseudoglandular phase (between 40 and 80 days of sheep gestation) when the bronchial tree and acini form. During the developmental progression to the canalicular phase (80–120 days of gestation) when differentiation into type I and II epithelial cells occurs, CFTR mRNA levels begin to decline, and remain low until birth. Larson et al. (28) have shown that overexpression of CFTR in the lungs of wild-type mice, at 15–16 days (corresponding to the late pseudoglandular and early canalicular developmental stages), advanced epithelial cell proliferation and differentiation. Furthermore, the same investigators suggested that transient expression of CFTR in CF mice following intramniotic injection of adeno-CFTR cDNA at 15–16 days of mouse gestation, corrected the lethal intestinal disease (29). The expression profile of CFTR in the developing sheep lung is consistent with data from the mouse that suggest a crucial role for CFTR in lung epithelial maturation. These observations may have important implications for current attempts to treat CF airway disease by postnatal gene therapy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Ovine tissue collection
An accurately timed sheep-breeding programme was established. Sheep breeds used were a cross between (ram) Poll Dorset and (ewes) Suffolk and Mule cross. Ewes were killed by anaesthetic overdose. Tissue was collected from fetuses and lambs at time points 52, 66, 74, 80, 83, 90, 94, 108, 129, 143 days of gestation, at term (147 days) and at 2 days and 2 weeks after birth. For most time points only one ewe was killed, however, at 74 days the ewe was carrying more than one fetus which were evaluated separately and at 2 days post-term two ewes were sacrificed. Tissues were snap-frozen in liquid nitrogen in cryotubes (Nunc) and stored in liquid nitrogen.
Extraction of RNA from ovine tissues
Tissue was homogenized in glass homogenizers and RNA was prepared by guanidinium thiocyanate extraction (30). Following ethanol precipitation samples were stored at –20°C. The RNA pellet was resuspended in nuclease-free water and OD260 determined. Volumes were adjusted to a concentration of 100–250 ng/µl before aliquoting and storage at –20°C.
RT–PCR for ovine CFTR and ENac
RT–PCRs were carried out as previously described by Chalkley and Harris (16) across the region encompassing exons 4 (4S3R, 5'-GGAATGCAGATGAGAATAGC-3') to 7 (A1L, 5'-GCTCCAAGAGAGTCATACCA-3') to ascertain whether alternative splicing of exons 6a and or 6b occurred. For semi-quantitative RT–PCR, CFTR cDNA synthesis was primed with 3' primer (C2L) 5'-TCTTCACTTATTTCCAAGCC-3'. cDNA was generated using Superscript (Life Technologies) at 42°C for 1 h, PCR was then performed with the 5' primer C2R (16) 5'-AGCAGTATACAAAGATGCTG-3' and C2L (94°C for 5 min, then 30 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min followed by a final cycle of extension at 72°C for 5 min). Primer concentrations were 10 ng/µl. Subunit c of sheep mitochondrial ATP synthase was coamplified as a control for total RNA using primers F1 and R1 at concentrations of 0.5 ng/µl (17). For semi-quantitative RT–PCR of ovine ENaC reactions were carried out as described for CFTR but using primers for the subunit of ENaC subunit. The 5' primer, 5'-CTGCTACACTTTCAACGACA-3', and the 3' primer, 5'-CTCCTTGATCATGCTCTCCT-3', were both used at 5 ng/µl, and primers for ovine ATP synthase were used at 0.5 ng/µl (annealing temperature of the PCR was 65°C and extension time was 5 min).
Real-time quantitative RT–PCR (TaqMan) using the ABI Prism 7700 sequence detection system (Applied Biosystems)
Primer and probe sequences were designed using the ovine CFTR cDNA sequence (GenBank accession no. U20418) and ABI PrimerExpress 1.0 software. The ovine CFTR 5' primer was located in exon 6a, 5'-AAGCCGGGTTAGGGAAAATG-3'. The 3' primer was located in exon 6b, 5'-TGCCTTAACTGATTGGATATTTTCAA-3'; the ovine CFTR TaqMan probe was 5'-CATTGATCTTTCCAGCTCTCTGATCTCTGTACTTCAT-3' and contained reporter dye FAM at the 5' end and quencher dye TAMRA at the 3' end. 18S rRNA primer and probes (5' primer, 5'-CGGCTACCACATCCAAGGAA-3'; 3' primer, 5'-GCTGGAATTACCGCGGCT-3'; and 5' VIC, 3' TAMRA modified probe 5'-TGCTGGCACCAGACTTGCCCTC-3') were supplied by Applied Biosystems.
A one-step RT–PCR approach was adopted for quantitative analysis. Independent assays were performed on each experimental sample to determine levels of both CFTR mRNA and 18S rRNA. Variations in RNA loading between samples were controlled by normalizing levels of CFTR mRNA to levels of 18S rRNA. CFTR RT–PCR assays (25 µl final volume) were performed using the TaqMan Gold RT–PCR Kit (Applied Biosystems) and contained 1x Buffer A, 7.5 mM MgCl2, 0.3 mM each of dATP, dCTP, dGTP and 0.6 mM dUTP, 0.025 U/µl AmpliTaq Gold, 0.25 U/µl MultiScribeRT, 0.2 U/µl RNase Inhibitor, 300 nM each of 5' and 3' primers and 100 nM probe, and included 5 µl of experimental total RNA template. 18S rRNA assays were identical in composition, except that reactions contained 5.5 mM MgCl2. Experimental total RNA samples were diluted to 10 ng/µl for the CFTR assays and to 0.01 ng/µl for the 18S rRNA assays. Reactions were carried out in MicroAmp Optical 96-well plates covered with MicroAmp Optical caps (Applied Biosystems). The TaqMan RT conditions were 48°C for 30 min followed by AmpliTaq Gold activation at 95°C for 10 min and 40 PCR cycles of 95°C for 15 s and 60°C for 1 min.
Data were analysed using ABI Sequence Detector software version 1.6.3. Relative standard curves for CFTR and 18S rRNA levels were generated from seven, sequential, 5-fold dilutions of a common arbitrary sample of lung RNA at 83 days gestation. The determined arbitrary values for CFTR were divided by the appropriate 18S rRNA value to give an expression ratio. This ratio was then expressed as a percentage of one chosen sample (the ‘calibrator’). For all experiments, total RNA at 83 days of gestation was used as the calibrator sample. Standards and experimental samples were assayed three times with each sample analysed in triplicate in each experiment. Reaction controls were performed which omitted RT and/or template RNA. Standard curves were fitted by linear regression and goodness of fit was reported as the coefficient of determination (R2).
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ACKNOWLEDGEMENTS |
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We thank Cliff Hanson and Frances Knight, David Smith, Sytse Henstra, Debbie Harrison, Andrew Rose and Ian Pringle for assistance. This work was funded by Vaincre La Mucoviscidose and the Cystic Fibrosis Trust. F.C.B.-C. was in receipt of a Medical Research Council postgraduate studentship.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Fax: +44 1865 222626; Email: aharris@molbiol.ox.ac.uk
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REFERENCES |
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M. A. Carey, J. W. Card, J. W. Voltz, D. R. Germolec, K. S. Korach, and D. C. Zeldin The impact of sex and sex hormones on lung physiology and disease: lessons from animal studies Am J Physiol Lung Cell Mol Physiol, August 1, 2007; 293(2): L272 - L278. [Abstract] [Full Text] [PDF]
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