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Human Molecular Genetics Pages 527-537

Co-ordinate regulation of the cystic fibrosis and multidrug resistance genes in cystic fibrosis knockout mice
Introduction
Results
Nature of Cftr mRNAs expressed inCftrtm1CAM mice
   Cftr expression inCftrtm1CAM mice
   Mdr1 expression inCftrtm1CAM mice
Discussion
   Cftr expression inCftrtm1CAM mice
   Mdr1expression in theCftrtm1CAM mice
Materials And Methods
   Mice and reagents
   In situ hybridisation
   Total RNA preparation, reverse transcription-PCR and RNase protection analysis
Acknowledgements
References

Co-ordinate regulation of the cystic fibrosis and multidrug resistance genes in cystic fibrosis knockout mice

Co-ordinate regulation of the cystic fibrosis and multidrug resistance genes in cystic fibrosis knockout mice Ann E. O. Trezise1,*, Rosemary Ratcliff2, Tim E. Hawkins2, Martin J. Evans2, Tom C. Freeman3, Pascale R. Romano1, Christopher F. Higgins1 and William H. Colledge4

1Nuffield Department of Clinical Biochemistry and Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital,Oxford OX3 9DU,UK,2Wellcome/CRC Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road,Cambridge CB2 1QR,UK,3Human Genetics Group, The Sanger Centre, Wellcome Trust Genome Campus,Hinxton, Cambs CB10 1SA,UK and 4Physiological Laboratory, University of Cambridge, Downing St,Cambridge CB2 3EG,UK

Received October 17, 1996;Revised and Accepted January 10, 1997

The cystic fibrosis (Cftr) and multidrug resistance (Mdr1) genes encode structurally similar proteins which are members of the ABC transporter superfamily. These genes exhibit complementary patterns of expressionin vivo, suggesting that the regulation of their expression may be co-ordinated. We have tested this hypothesisin vivo by examiningCftr and Mdr1 expression in cystic fibrosis knockout transgenic mice (Cftrtm1CAM). Cftr mRNA expression inCftrtm1CAM/Cftrtm1CAM mice was 4-fold reduced in the intestine, as compared with littermate wild-type mice. All otherCftrtm1CAM/Cftrtm1CAM mouse tissues examined showed similar reductions inCftr expression. In contrast, we observed a 4-fold increase in Mdr1 mRNA expression in the intestines of neonatal and 3- to 4-week-oldCftrtm1CAM/Cftrtm1CAM mice, as compared with age-matched +/+ mice, and an intermediate level of Mdr1 mRNA in heterozygousCftrtm1CAM mice. In 10-week-old,Cftrtm1CAM/Cftrtm1CAM mice and in contrast to the younger mice, Mdr1 mRNA expression was reduced, by 3-fold. The expression of two control genes,Pgk-1 and Mdr2, was similar in all genotypes, suggesting that the changes in Mdr1 mRNA levels observed in theCftrtm1CAM/Cftrtm1CAM mice are specific to the loss ofCftr expression and /or function. These data provide further evidence supporting the hypothesis that the regulationCftr and Mdr1 expression isco-ordinatedin vivo, and that this co-ordinate regulation is influenced by temporal factors.

INTRODUCTION

Mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) cause cystic fibrosis (1 -3 ). Cystic fibrosis affects a number of organ systems, including the respiratory, digestive and reproductive systems (4 ), and its underlying cause is the aberrant expression and /or function of a cAMP-regulated chloride channel encoded byCFTR (5 -7 ). As well as functioning as a chloride channel, CFTR also regulates other ion channels (8 ,9 ).

The primary structure of CFTR places it in the ABC transporter superfamily (2 ,10 ). The P-glycoproteins, encoded by theMDR1 and MDR2 genes in humans and by themdr1a,Mdr1b and Mdr2 genes in rodents, are also members of the ABC transporter superfamily (reviewed in11 ). The P-glycoproteins encoded by humanMDR1 and mouseMdr1a and Mdr1b can confer multidrug resistance by transporting chemotherapeutic drugs out of cells. In addition to their drug transport function, human MDR1 and mouse Mdr1a modulate the activation of cell swelling-activated chloride channels (12 -14 ). Thus, theCftr and Mdr1 genes are structurally related, sharing a common domain organisation, and functionally related, acting as regulators of heterologous ion channels.

We have shown previously thatCftr and Mdr1a and /or 1b (collectively referred to asMdr1) exhibit complementary patterns of expressionin vivo (5 ). That is, in cells which expressCftr,Mdr1 expression cannot be detected, and vice versa. Furthermore, during development and differentiation, certain cells switch expression from one gene to the other. For example,Cftr is expressed in the epithelia of the intestinal crypts whileMdr1 is expressed in the villous epithelia. As the epithelial cells migrate across the crypt-villous boundary,Cftr expression is reduced to undetectable levels whileMdr1 expression is switched on. Similarly,Cftr is expressed in the uterine epithelia during the oestrous cycle; then at the onset of pregnancy these cells turn off expression ofCftr and switch onMdr1expression. These findings led us to propose that the regulation ofCftr and Mdr1 expression may be co-ordinated (15 ). A number ofin vitro studies also suggest co-ordinate regulation ofCftr and Mdr1 expression. Galactose-induced differentiation of HT-29 cells (human intestinal cell line) is accompanied by opposite shifts inCFTR and MDR1 expression (16 ). Similarly, colchicine induction ofMDR1 expression is accompanied by an 80% decrease inCFTR expression. Both effects are reversed following removal of the drug (17 ).

The creation of cystic fibrosis knockout transgenic mice has allowed us to test the co-ordinate regulation hypothesisin vivo. Cystic fibrosis knockout transgenic mice have been constructed, independently, by several groups (18 -23 ). We have used RNAin situ hybridisation to examineCftr and Mdr1 expression in theCftrtm1CAM null mice (19 ). These mice were created using a replacement targeting strategy and have a severe cystic fibrosis phenotype with most animals succumbing to intestinal blockages early in life. No Cftr-related chloride conductances can be detected in the intestinal, tracheal or pancreatic duct epithelia ofCftrtm1CAM/Cftrtm1CAM mice (18 ,19 ,24 ). It is possible that in the absence ofCftr expression/function, the cellular response may be to activate other chloride channels by increasing the expression of genes encoding chloride channel regulators, such as Mdr1. The hypothesis of co-ordinate regulation ofCftr and Mdr1 expression predicts that an alteration inCftr expression should lead to changes inMdr1 expression. Here we show that the level of Cftr mRNA is greatly reduced in all tissues examined inCftrtm1CAM/Cftrtm1CAM mice, as compared with wild-type mice. This decrease in Cftr mRNA levels is accompanied by altered Mdr1 mRNA expression, providing supporting evidence for the hypothesis that expression ofCftr and Mdr1is co-ordinately regulatedin vivo.

RESULTS

Nature of Cftr mRNAs expressed inCftrtm1CAM mice

We investigated the nature of the mRNAs transcribed from theCftrtm1CAM locus to assess the effectiveness of the transcription termination signals inserted in exon 10. Figure1 shows the structure of part of theCftrtm1CAMlocus surrounding the targeted disruption in exon 10, and a number of possible RNAs that could be transcribed from this gene. The Cftr promoter has not been altered in theCftrtm1CAM mice and , therefore, transcription initiation is not expected to be affected by the gene disruption. If RNAs initiated at the Cftr promoter are terminated at the targeted disruption, as predicted, then the transcript shown in Figure1 a would be produced. The constitutive Pgk-1 promoter, which was introduced at the targeted disruption inCftr exon 10, is also expected to initiate transcription. If these transcripts are terminated at the Pgk-1 poly(A)+/transcription termination signals then they would only contain aHPRTR coding sequence (transcripts not shown). However, if the Pgk-1 poly(A)+/transcription termination signals are not 100% efficient, then two other RNAs containing theCftr coding sequence are predicted. One of these is initiated from the Pgk-1 promoter (Fig.1 b), and the other from the endogenous Cftr promoter (Fig.1 c). It is also possible that a Cftr mRNA spliced directly from exon 9 to exon 11 may be produced (Fig.1 d), although this would result in a frame-shift mutation and the introduction of downstream, in-frame translation stop codons. Cryptic splice sites in and around exon 10 and the targeted mutation might exist, although there is no evidence for such sites being used in theCftrtm1CAM mice. None of the predicted Cftr mRNAs shown in Figure1 can encode a functional Cftr protein, and electrophysiological analysis of theCftrtm1CAM mice demonstrates a null Cftr phenotype (18 ,19 ,24 ).


Figure 1. Diagram of exons 9-11 of the disruptedCftr locus and mRNAs predicted to be expressed inCftrtm1CAM mice. The RNA shown in (a) would be predicted if RNA transcription initiated normally at theCftr promoter and was terminated at the introduced mini-gene transcription termination signal. The RNAs shown in (b) and (c) would be predicted if there is read-through of the introduced mini-gene transcription termination signal. The RNA shown in (b) is initiated at the introduced Pgk-1 promoter and the RNA shown in (c) is initiated at the Cftr promoter.

We used reverse transcription-polymerase chain reaction (RT-PCR) and RNase protection analyses to investigate the nature of Cftr transcripts expressed from theCftrtm1CAMlocus. In tissues that usually expressCftr, all the possible Cftr mRNAs shown in Figure1 would be anticipated, whereas in tissues that do not usually expressCftr only the Cftr mRNA shown in Figure1 b is predicted. In the RT-PCR, two Cftr fragments were amplified, one from exons 4-6 upstream of the targeted disruption (Fig.2 , lanes `u'), and the other from exons 11-13 downstream of the disruption (Fig.2 , lanes `d'). Amplification of [beta]-actin mRNA (Fig.2 , lanes `a') was used as a positive control. InCftrtm1CAM/Cftrtm1CAM mice, the upstream primers generated a 480 bp product from total RNA extracted from lung, kidney, uterus, testis, small intestine, colon, brain and stomach (Fig.2 , lanes `u' and data not shown). However, no upstream product could be amplified from spleen, liver, heart and skeletal muscle RNA (Fig.2 , lanes `u' and data not shown). So, amplification of the upstream PCR product coincides accurately with the known tissue distribution of Cftr mRNA (25 ,26 ). In+/+ mice, the downstream PCR product (375 bp) could only be amplified from RNA prepared from tissues known to expressCftr (Fig.2 , lanes `d' and data not shown), whereas in theCftrtm1CAM/Cftrtm1CAM mice, the downstream Cftr primers amplified a 375 bp PCR product from RNA isolated from all tissues examined, including those where no upstream PCR product was observed (Fig.2 , lanes `d' and data not shown). Presumably, this is due to transcription initiation from the ubiquitously expressed Pgk-1 promoter (contained within the targeting construct) and some read-through of the transcription termination signals of theHPRTR mini-gene (see Fig.1 b).


Figure 2. Reverse transcription-PCR analysis ofCftr expression in stomach and skeletal muscle ofCftrtm1CAM mice. Lane a shows the products of a control amplification of [beta]-actin mRNA sequences. Lane u shows the products of an amplification of exons 4-6 of Cftr mRNA sequences, upstream of the targeted disruption in exon 10. Lane d shows the products of an amplification of exons 11-13 of Cftr mRNA sequences downstream of the targeted disruption in exon 10. Lane M shows size st and ards. The sizes (bp) of the amplified fragments are shown on the left.

To assess the extent of transcriptional read-through of theHPRTR mini-gene, we used RNase protection analysis with a probe specific forCftr exon 10 and spanning the targeted disruption. A 191 bp RNA fragment from this probe (plasmid pE16) will be protected by wild-type Cftr mRNA and this was observed with RNA isolated from testis, kidney and intestine of+/+ and +/Cftrtm1CAM animals, but notCftrtm1CAM/Cftrtm1CAM mice (Fig.3 A). A 71 bp fragment was protected by RNA isolated from the testis ofCftrtm1CAM/Cftrtm1CAM mice (Fig.3 A, lane -/- T and data not shown), the size predicted for RNA containing the 5' region ofCftr exon 10 up to the insertedHPRTR mini-gene. The decreased signal from this 71 bp fragment, compared with the 191 bp fragment in the+/+ mice, is due in part to the decreased size of the protected fragment, but also suggests that the amount of truncated Cftr mRNA (Fig.1 a) was greatly reduced. Inability to detect this 71 bp fragment in the kidney and intestine, which express similar Cftr mRNA levels to the testes, suggests differential stability of the truncated Cftr mRNA (Fig.1 a) in different tissues. A second probe, containing the 3' region of exon 10 (generated from plasmid p987), would be predicted to generate a 122 bp protected fragment when hybridised either to wild-type Cftr mRNA or to the fused HPRTR-Cftr transcripts resulting from read-through of the poly(A)+ signal sequence (Fig.1 b and c). A 122 bp fragment was protected by RNA from the testis, kidney and intestine of+/+ and +/Cftrtm1CAM animals, but notCftrtm1CAM/Cftrtm1CAM animals (Fig.3 B), indicating that the only downstream sequences detected by RNase protection were from wild-type Cftr mRNA. InCftrtm1CAM/Cftrtm1CAM mice, the detection of Cftr mRNAs containing sequences downstream of the targeted interruption by RT-PCR, but not by RNase protection, suggests that transcriptional read-through of the Pgk-1 termination signal is very low. This analysis establishes that the targeted disruption inCftrtm1CAM exon 10 effectively terminates the majority of transcription at this point.

Cftr expression inCftrtm1CAM mice

RNAin situ hybridisation was used to study the localisation and level of Cftr mRNA expression inCftrtm1CAM mice at three different stages of development: neonatal (1 day), suckling-weaning transition (3-4 weeks) and adult (10 weeks). We used two probes to examineCftr expression, one upstream of the targeted disruption of theCftr locus and the other downstream. The upstream Cftr probe hybridised to exons 3-5 (bp 309-695), and the downstream probe recognised exons 11-13 (bp 1771-2112 according to ref.27 ). The RT-PCR and RNase protection analyses have shown that the majority of the Cftr mRNA expressed in theCftrtm1CAM/Cftrtm1CAM mice was initiated at the Cftr promoter and terminated at the targeted disruption inCftr exon 10 (see Fig.1 a).So, the hybridisation signal detected with the upstream Cftr probe (see Fig.4 ) indicates the localisation and level of mRNAs initiated at the Cftr promoter (Fig.1 a, c and d).


Figure 3. RNase protection analysis ofCftr expression inCftrtm1CAM mice.Cftr expression was analysed in testis (T), kidney (K), intestine (I), heart (H) and skeletal muscle (M). (A) RNase protection using a probe specific for Cftr exon 10 and spanning the targeted interruption. Arrows indicate specifically protected fragments at 191 and 71 bp. (B) RNase protection using a probe to the 3' region of Cftr exon 10. The arrow indicates a specifically protected fragment at 122 bp.


Figure 4. Cftr mRNA expression in 1-day-old,Cftrtm1CAM mice. (A,D and G) Brightfield images of cryostat sections of the small intestine that have been hybridised to the antisense, upstream Cftr probe. Arrows indicate intestinal intervillous epithelia, from which the intestinal crypts will develop during post-natal weeks 1 and 2. (B,E and H) Darkfield images of the same sections allowing visualisation of the hybridisation signal (white dots). The intensity of the hybridisation signal gives an indication of the amount of mRNA present. (C,F and I) Darkfield images of consecutive sections hybridised with the corresponding sense str and probe, which serves as a negative control. The sections in (A), (B) and (C) are from a+/+ mouse, those in (D), (E) and (F) from a+/Cftrtm1CAM mouse and those in (G), (H) and (I) from aCftrtm1CAM/Cftrtm1CAM mouse. The size bar equals 200 µm.

In 1-day-old,+/+ mice, the upstream Cftr probe readily detected Cftr mRNA in the intestinal intervillous epithelia from which the intestinal crypts develop during postnatal weeks 1 and 2 (see Fig.4 A and B). In contrast, very little Cftr mRNA could be detected in the intestines ofCftrtm1CAM/Cftrtm1CAM mice (Fig.4 G and H); in+/Cftrtm1CAM mice an intermediate amount of Cftr mRNA was observed (Fig.4 D and E). At all times, the experimenters performing the RNAin situ hybridisations were blinded with respect to the genotypes of the animals examined. Using RNAin situ hybridisation to examine Cftr mRNA levels, the genotypes ofCftrtm1CAM mice were identified correctly in 28/30 cases and all theCftrtm1CAM/Cftrtm1CAM mice (nine) were identified correctly.

Relative levels of mRNA expression were determined objectively by densitometric analysis of X-ray film contact sheets made from slides prior to autoradiography (see Materials and Methods). This analysis showed that the hybridisation signal in 1-day-old,+/+ mice was clearly detectable, while the signal inCftrtm1CAM/Cftrtm1CAM mice was not above background level.

We also examinedCftr expression in the intestines of 3- to 4-week-old (suckling-weaning transition) and 10-week-old (adult) mice. The results of these experiments are summarised in Table1 . Compared with+/+ mice, the upstream Cftr probe detected reduced, but still clearly detectable levels of Cftr mRNA in the submucosal (Brunner's) gl and s of the intestine of 3- to 4-week-oldCftrtm1CAM/Cftrtm1CAM mice. However, the downstream probe detected very little Cftr mRNA in the Brunner's gl and s ofCftrtm1CAM/ Cftrtm1CAM mice, much less than the upstream probe. In wild-type mice, both probes detected approximately equivalent amounts of Cftr mRNA. Identical results were obtained from the 10-week-oldCftrtm1CAM mice. These data suggest the presence of significant levels of the truncated Cftr mRNA (Fig.1 a) in the Brunner's gl and s ofCftrtm1CAM/Cftrtm1CAM mice, and a low level of read-through of the introduced transcription termination signals.Cftr expression in the intestinal crypt epithelia of 3- to 4-week-old and 10-week-old mice was similar to that observed in neonatal mice. Both upstream and downstream probes detected 4-fold reduced Cftr mRNA expression in the ileal crypts of 10-week-oldCftrtm1CAM/Cftrtm1CAM mice, as compared with age-matched+/+ mice.

The salivary gl and s are also a major site ofCftr expression (25 ,28 ). Similar levels of Cftr mRNA were detected with both the Cftr probes in the intra- and interlobular ducts of the salivary gl and s of neonatal,+/+ mice (data summarised in Table1 ). InCftrtm1CAM/Cftrtm1CAM mice, the upstream probe detected Cftr mRNA in the intra- and interlobular salivary gl and ducts, while the downstream Cftr probe did not detect Cftr mRNA expression. This pattern of expression is similar to that seen in the intestinal Brunner's gl and s of theCftrtm1CAM mice, and again suggests the presence of the truncated Cftr mRNA (Fig.1 a) in the salivary gl and ducts ofCftrtm1CAM/Cftrtm1CAM mice.

Mdr1 expression inCftrtm1CAM mice


Figure 5.Mdr1 expression in the intestinal crypt (c) and villous (v) epithelia of 3- to 4-week-oldCftrtm1CAM mice. (A,D and G) Brightfield images of sections hybridised to an antisense Mdr1 probe; (B,E and H) show darkfield images of these sections. (C,F and I) Darkfield images of consecutive sections hybridised to the sense Mdr1 probe.Mdr1 expression in+/+,+/Cftrtm1CAM and Cftrtm1CAM/Cftrtm1CAM mice is shown in (A-C), (D-F) and (G-I), respectively. The size bar equals 200 µm.


Figure 6.Mdr1 expression in the ileum of 10-week-oldCftrtm1CAM mice. (A and B)Mdr1 expression in the villous (V), but not the crypt epithelia (C) of a+/+ mouse. (C and D)Mdr1 expression is greatly reduced in the ileum of aCftrtm1CAM/Cftrtm1CAM mouse. (A) and (C) show brightfield images of sections hybridised to the antisense Mdr1 probe while (B) and (D) show the corresponding darkfield images. The size bar equals 100 µm.Co-ordinate regulation ofCftr and Mdr1 would predict that alterations in the expression of one gene would lead to subsequent changes in the expression of the other. In rodents, the homologue of the humanMDR1 gene has been duplicated and the two mouse genes are designatedMdr1a and Mdr1b (also known asMdr3 and Mdr1, respectively, ref.11 ). Since we are interested in combinedMdr1a and Mdr1b expression in theCftrtm1CAM mice, we used a probe which hybridised to bothMdr1a and Mdr1bsequences. We will refer to mouseMdr1 expression throughout to denote combinedMdr1a and /orMdr1b expression. The sequences of exon 6 of mouseMdr1a and Mdr1b contain only five differences out of 192 bp, so exon 6 of mouseMdr1b was used to examineMdr1 expression. Southern blotting analysis demonstrated that this probe recognised bothMdr1a and Mdr1b, but notMdr2 (data not shown).

Table 1. Cftr mRNA expression in the intestines and salivary gl and s of wild-type (+/+) and Cftrtm1CAM/Cftrtm1CAM mice
Tissue and age

 Genotype

 Upstream Cftr probe

 Downstream Cftr probe
Intestinal Brunner's gl and s

 +/+

 ++++

 ++++
(weaning and adult mice)

 Cftrtm1CAM/Cftrtm1CAM

 +++

 +
Intestinal crypt epithelia

 +/+

 +++

 +++
(weaning and adult mice)

 Cftrtm1CAM/Cftrtm1CAM

 + (4-fold reduced)

 + (4-fold reduced)
Salivary gl and ducts

 +/+

 +++

 +++
(neonates)

 Cftrtm1CAM/Cftrtm1CAM

 +++

 -
Relative signal intensities: ++++ = high; +++ = moderate; ++ = low; + = just detectable; - = not detectable.Age groups: neonatal = 1-3 days; weaning = 3-4 weeks; adult = 10 weeks.

Reduced levels of Cftr mRNA expression were observed in the intestines of 1-day-old and 3- to 4-week-oldCftrtm1CAM/ Cftrtm1CAM mice (Fig.4 ). Therefore,Mdr1 expression was studied in a series of consecutive sections. The cellular patterns ofCftr and Mdr1 expression observed in 3- to 4-week-old mice were similar to those found in the 1-day-old mice. In+/+ mice of both age groups, low levels of Mdr1 mRNA were observed in the intestinal intervillous and villous epithelia (Fig.5 A and B). The expression ofMdr1 in both intervillous and villous epithelial cells differed from the pattern of expression in adult+/+ mice where expression was restricted to the villous epithelia (ref.15 , and see Fig.6 ). In 1-day-old and 3- to 4-week-oldCftrtm1CAM/Cftrtm1CAM mice, Mdr1 mRNA expression was also observed in intervillous and villous epithelia, but the level ofMdr1 expression was increased 4-fold compared with+/+ mice (Fig.5 G and H). We also examined intestinalMdr1 expression in 3- to 4-week-old heterozygous mice. The cellular distribution of Mdr1 mRNA was identical in all three mouse genotypes, and an intermediate level ofMdr1 expression was observed in the+/Cftrtm1CAM mice as compared with+/+ and Cftrtm1CAM/Cftrtm1CAM mice (Fig.5 , compare E with B and H, respectively).

To determine whether the changes inMdr1 expression observed in 1-day-old and 3- to 4-week-oldCftrtm1CAM/ Cfttm1CAM mice were affected by intestinal development, we also examinedMdr1 expression in 10-week-old (adult)Cftrtm1CAM mice. In adult+/+ mice,Mdr1 expression was restricted to the duodenal and ileal villous epithelia with no evidence of expression in the crypt epithelia or Brunner's gl and s (Fig.6 A and B and data not shown), typical of the mature pattern of intestinalMdr1 expression (15 ). Comparison of Mdr1 mRNA expression in all three age groups of+/+ mice revealed a gradual contraction in expression from the intervillous/crypt and villous epithelia seen in mice up to the age of weaning, to the villous epithelia of adult mice. There was also an increase in the level ofMdr1 expression in the villous epithelia after weaning was complete. In contrast to our observations in the youngerCftrtm1CAM/Cftrtm1CAM mice, we observed a marked decrease in Mdr1 mRNA expression in the duodenal and ileal villous epithelia of 10-week-oldCftrtm1CAM/Cftrtm1CAM mice (Fig.6 C and D), as compared with+/+ mice (Fig.6 A and B). The levels of Mdr1 mRNA were 3-fold less in the small intestines of 10-week-oldCftrtm1CAM/Cftrtm1CAM mice, compared with+/+ mice. Our observations suggest that the transition through weaning results in Mdr1 mRNA expression changing from overexpression in immatureCftrtm1CAM/Cftrtm1CAM intestine, to underexpression in adultCftrtm1CAM/Cftrtm1CAM intestine. This is in contrast to the developmental changes inMdr1 expression in+/+ intestine, where an overall increase in Mdr1 mRNA levels was observed after weaning.

To establish that the changes inMdr1 expression observed in theCftrtm1CAM/Cftrtm1CAM mice are specific to the loss ofCftr expression and /or function, and not a result of a general alteration in expression of all genes in theCftrtm1CAM/Cftrtm1CAM mice, we examinedMdr2 and Pgk-1expression in 3- to 4-week-old and 10-week-oldCftrtm1CAM mice (Fig.7 ). The mouse Mdr2 probe recognisedMdr2 exon 21, and Southern blotting showed that this probe hybridised specifically to mouseMdr2, but not toMdr1a orMdr1b (data not shown). A human PGK-1 probe, which cross-hybridises with mouse Pgk-1 sequences (29 ), was used to assess mousePgk-1 expression.


Figure 7.Pgk-1 expression in the duodenum (A-H) and ileum (I-P) of 3- to 4-week-old, and 10-week-oldCftrtm1CAM mice. The intestinal crypts (c), villi (v) and Brunner's gl and s (b) are marked. (A,C,E,G,I,K,M and O) Brightfield images of sections hybridised to the PGK-1 probe. (B,D,F,H,J,L,N and P) Darkfield images of these same sections. Sections in (A), (B), (E), (F), (I), (J), (M) and (N) are from a+/+ mouse and sections in (C), (D), (G), (H), (K), (L), (O) and (P) are from aCftrtm1CAM/Cftrtm1CAM mouse. The sections in (A-D) and (I-L) are from 3- to 4-week-old mice and the sections in (E-H) and (M-P) are from 10-week old-mice. The size bar equals 200 µm.

Mdr2 is not normally expressed in the intestine (30 ) and no transcripts were detected in the intestines of either+/+ orCftrtm1CAM/Cftrtm1CAM mice (data not shown).Pgk-1 was found to be expressed in the duodenal and ileal crypt and villous epithelia (Fig.7 ). The highest level of Pgk-1 mRNA was observed in the crypt epithelia, with a decreasing gradient of expression along the crypt-villous axis. Pgk-1 mRNA was not detected in the Brunner's gl and s of the intestine (Fig.7 A-H). In both age groups ofCftrtm1CAM mice, the level and cellular distribution of expression of Pgk-1 mRNA in the intestine was similar in+/+ and Cftrtm1CAM/Cftrtm1CAM mice (Fig.7 ). Therefore, we conclude that the changes inMdr1 expression observed in theCftrtm1CAM/Cftrtm1CAM mice are specific to the loss ofCftr expression and /or function.

DISCUSSION

Cftr expression inCftrtm1CAM mice

TheCftrtm1CAM mice were produced by a replacement targeting event in which anHPRTR mini-gene was inserted into exon 10 of theCftr locus. RT-PCR, RNase protection and RNAin situ hybridisation data suggest the level of read-through of theHPRTR mini-gene is low and that the majority of the Cftr mRNA detected in theCftrtm1CAM/Cftrtm1CAM mice is the truncated Cftr mRNA shown in Figure1 a. We were able to detect the truncated Cftr mRNA (Fig.1 a) in the Brunner's gl and s and salivary gl and ducts, but not in the intestinal crypt epithelia of theCftrtm1CAM/ Cftrtm1CAM mice.Cftr is expressed at similar levels in the salivary gl and ducts and intestinal crypt epithelia of wild-type mice. This suggests tissue-specific differences in the stability of the truncated Cftr mRNA, and that the truncated mRNA is less stable than wild-type Cftr mRNA in crypt epithelia. This is not surprising since exons 11-24 and the 3' untranslated region (3' UTR) have been deleted, and the 3' UTR often contains sequence motifs important in determining mRNA stability (31 ). This finding suggests that the 3' UTR may also be important for Cftr mRNA stability in some cell types. It is also possible that the alterations in Cftr mRNA levels may be due to changes in the rate of transcription initiation from theCftrtm1CAM promoter, and that these changes in transcription are further complicated by the altered stability of the mutant Cftr mRNAs. As the disruption of theCftrtm1CAM locus is contained entirely within exon 10 it is unlikely that this event alters the rate of transcription initiation from theCftrtm1CAM promoter, although this has not been demonstrated formally. Overall, these studies show that Cftr mRNA levels are greatly reduced in theCftrtm1CAM/Cftrtm1CAM mice and any Cftr mRNA expressed is largely terminated at the targeted interruption.

Mdr1expression in theCftrtm1CAM mice

The data presented here show thatMdr1 expression is developmentally regulated in the intestines of wild-type mice. In neonatal and suckling-weaning mice,Mdr1 was expressed at low levels throughout the intervillous/crypt and villous epithelia, while in adult mice the level ofMdr1 expression was increased and restricted to the villous epithelia. The transition through weaning is a crucial stage of intestinal development and is known to produce changes in the expression of a number of genes that play important roles in intestinal physiology (32 ,33 ). The developmental regulation ofMdr1 expression in the intestine is consistent with such a role for Mdr1 in normal intestinal function.

We have shown previously thatCftr and Mdr1 exhibit complementary patterns of expressionin vivo, suggesting that the regulation of expression of the two genes may be co-ordinated (15 ). The results of this study provide further evidence for the co-ordinate regulation ofCftr and Mdr1 expressionin vivo.

In the intestinal epithelia of neonatal (1-day-old) and suckling-weaning (3- to 4-week-old)Cftrtm1CAM/Cftrtm1CAM mice, Mdr1mRNAexpression was increased compared with wild-type mice, while in adultCftrtm1CAM/Cftrtm1CAM miceMdr1 was underexpressed. The expression of two control genes was not altered in theCftrtm1CAM/Cftrtm1CAM mice, indicating that the observed changes inMdr1 expression were specific to the loss ofCftr expression/function. We also observed intermediate levels ofMdr1 expression in+/Cftrtm1CAM mice, suggesting that alterations in Mdr1 mRNA levels are regulated in response to decreasedCftr expression and /or function. Furthermore, as the+/Cftrtm1CAM mice are phenotypically wild-type (19 ), this suggests that the changes inMdr1 expression are not due to a general stress response associated with the severe intestinal phenotype of theCftrtm1CAM/Cftrtm1CAM mice. The changes inMdr1 expression observed in theCftrtm1CAM/Cftrtm1CAM mice are also influenced by the developmental stage of the intestine. The transition through weaning results inMdr1 expression inCftrtm1CAM/Cftrtm1CAM intestines changing from overexpression to underexpression, suggesting that the co-ordinate regulation ofCftr and Mdr1 expression is also subject to temporal regulation associated with weaning.

As only a small percentage ofCftrtm1CAM/Cftrtm1CAM mice survive to 10 weeks, it is possible that these mice may represent a sub-population, and that down-regulation ofMdr1 expression in 10-week-oldCftrtm1CAM/Cftrtm1CAM mice does not represent the true pattern ofMdr1 expression in the general population of adultCftrtm1CAM mice. If this were the case then we would expect to see some immatureCftrtm1CAM/Cftrtm1CAM mice showing underexpression ofMdr1 in their intestines. We analysedMdr1 expression in nine neonatal and suckling-weaningCftrtm1CAM/ Cftrtm1CAM mice and always observed increased intestinalMdr1 expression. For similar reasons, we suggest that the modifier locus, which modulates the severity of the cystic fibrosis phenotype in theCftrtm1HSC mice (22 ), is unlikely to have an impact onMdr1 expression in theCftrtm1CAM mice.

This work, together with our previous studies of the expression ofCftr and Mdr1, suggests that the expression of both these genes is subject to spatial, hormonal, temporal and co-ordinate regulation (15 ,26 ,34 ). A number of possible mechanisms underlying the co-ordinated regulation ofCftr and Mdr1 can be envisaged. It is possible that a reduction or loss of Cftr function influences Mdr1 mRNA expression. This model is consistent with the observation of increasedMdr1 expression in theCftrtm1CAM/ Cftrtm1CAM mice, which express no functional Cftr protein, and the intermediate increase in Mdr1 mRNA levels in+/Cftrtm1CAM mice. However, it is equally possible that some other aspect ofCftr expression influencesMdr1 expression, for example mRNA levels. Any proposed model will also have to take into account the interaction between mechanisms directing the co-ordinate regulation ofCftr and Mdr1 and the temporal regulation ofMdr1.

MATERIALS AND METHODS

Mice and reagents

The population of cystic fibrosis knockout mice was created and bred at the Wellcome/CRC Institute in Cambridge (Cftrtm1CAM mice). The genotypes of the mice were established by PCR and Southern blot analysis as described previously (18 ,19 )

In situ hybridisation

Animals were sacrificed by a lethal injection of anaesthetic and tissues fixed by whole body perfusion with 4% paraformaldehyde.In situ hybridisation was carried out essentially as described previously (26 ). Cryostat sections (10 µm) were hybridised to35S-labelled, single-str and ed RNA probes. The antisense str and probe will hybridise to the mRNA and identify cells expressing the gene of interest. The sense str and probe serves as a negative control.

The upstream and downstream mouse Cftr probes were generated by PCR and have been described previously (26 ). The mouse Mdr1b exon 6 probe was produced by PCR amplification from the mouse Mdr1b cDNA (a generous gift from Dr P. Gros). The sequence of the PCR DNA fragment exactly matched the published mouseMdr1b sequence, and corresponded to nucleotides 446-635 (35 ). The mouse Mdr2 exon 21 probe was generated by PCR from mouse genomic DNA. The sequence of the mouse Mdr2 exon 21 probe exactly matched the published mouseMdr2 sequence and corresponded to positions 2663-2864 (36 ). The human PGK-1 probe, which cross-hybridises with mouse Pgk-1 sequences, corresponded to 68 bp of intron 2 and 121 bp of exon 3 of humanPGK-1. The plasmid containing the human PGK-1 insert was kindly provided by Dr J. Firth (29 ). All DNA fragments used asin situ hybridisation probes were subcloned into pSK+ (Bluescript: Stratagene) to allow thein vitro transcription of the insert using T3 and T7 RNA polymerases.

In situ hybridisation signal intensities were estimated by densitometric analysis of contact sheets ofin situ hybridisation slides made prior to dipping the slides in photographic emulsion. Contact sheets were obtained using Amersham [beta]-max film and hybridisation signal intensities quantitated using an Seescan densitometer (Cambridge, UK). Hybridisation signals from a given probe were quantitated by averaging readings from a minimum of three sections per probe per animal. In the case of+/+, 1-day-old animals, measurements from two animals were averaged to a give a final measure of the level of signal from a given probe. In all other cases, measurements were averaged from three animals.

Total RNA preparation, reverse transcription-PCR and RNase protection analysis

Total RNA was prepared using either the acid guanidinium thiocyanate phenol method (37 ), or by lithium chloride urea extraction (38 ).

RT-PCR was carried out according to (39 ). Two pairs of primers were used. One pair amplified a fragment of 480 bp, containing exons 4-6 of murineCftr upstream of the targeted interruption in exon 10 of theCftrtm1CAM mice. The other pair of primers amplified a fragment of 375 bp, corresponding to exons 11-13 downstream of the targeted disruption. A pair of primers which amplified a 500 bp fragment of [beta]-actin cDNA (40 ) were used as a positive control.

The primers forCftr exons 4-6 were: SM3, 5'-tccag cctgt cttgc tagga agaat-3' and SM4, 5'-cattg atctt tgcag ctctt tgatc-3'. The primers forCftr exons 11-13 were: X11F, 5'-GACAT CACCA AGTTT GCAGA A-3' and SDCF4, 5'-AAACT GGTCA AAAGT ATCAT AC-3'. The primers for [beta]-actin were: Actin 1, 5'-ATGGA TGACG ATATC GCTG-3' and Actin 2, 5'-ACCTG ACAGA CTACC TCAT-3'.

RNase protection assays were performed using st and ard techniques (41 ) except that tRNA was omitted from the reaction. After digestion with RNase A and T1, the digestion products were electrophoresed on denaturing, 10% polyacrylamide gels and protected fragments were visualised by autoradiography. The plasmid pE16 was generated by ligation of a 791 bpSau3AI-NsiI fragment, containing the whole of the mouseCftr exon 10 as well as some flanking intron sequence, into theBamHI-PstI sites of pT7/T3[alpha]-19 (Gibco BRL). Plasmid p987 was generated by ligation of a 906 bpBglII-NsiI fragment derived from pCFB-HPRT (18 ) into theBamHI-PstI sites of pT7/T3[alpha]-19. p987 therefore contains only that part of mouse exon 10 which is downstream of theHPRT insertion plus some flanking intron 10 sequence. Riboprobes were generated from these plasmids for RNase protection assays using T7 RNA polymerase according to the manufacturer's instructions.

ACKNOWLEDGEMENTS

We thank Drs Kevin Spring, Lis Mudd, Simon Hardy, Jay Hinton and Miss Nina Birchall for advice and discussions. A.E.O.T. was a Beit Memorial Research Fellow during the course of this research and C.F.H. is a Howard Hughes International Research Scholar. This research was supported by the Wellcome Trust, the Henry Smith Foundation, the Cystic Fibrosis Research Trust, the Imperial Cancer Research Fund and the Medical Research Council.

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*To whom correspondence should be addressed

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