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First published online 26 January 2005
doi: 10.1242/dev.01662

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E-cadherin intron 2 contains cis-regulatory elements essential for gene expression

¹ Department of Molecular Embryology, Max-Planck Institute of Immunobiology, Stuebeweg 51, D-79108 Freiburg, Germany
² Andreas Hecht, Institute of Molecular Medicine and Cell Science, University of Freiburg, Stefan-Meier-Strasse 17, D-79104 Freiburg, Germany

View larger version (46K): [in a new window]   Fig. 1. Generation of ES cells with targeted floxed E-cadherin intron 2. (A) Schematic representation of the E-cadherin locus (drawn to scale, 1). Exons are represented by vertical black bars, and nucleotide positions are given with respect to the transcription start site (+1). The locus was targeted with vector TV1 (2) and subsequently with TV2 (3), with detailed analysis after each step, finally resulting in the double-targeted allele (4) to delete intron 2 by Cre recombinase expression (5). For additional negative selection, a herpes simplex virus thymidine kinase gene (HSV-tk) was integrated in TV1 and betageo was fused in-frame to the E-cadherin start codon. In TV2 a hygromycin resistance cassette (hygr) under the control of the phosphoglycerol kinase promoter (PGK) was inserted in reverse orientation 5' of exon 3. Promoter (P), exons (E1, E2, etc.), loxP sites (red triangles), FRT sites (blue triangles), polyadenylation signals (striped boxes), transcription start sites (horizontal arrows), used restriction sites and probes (horizontal red bars) are given. The expected fragments of the Southern blot analysis for the homologous recombination of TV1 with probe a are indicated by green bars, and those for TV2 with probe f by blue bars. If both events occur at the same allele (in cis), a 46 kb fragment is expected after digestion with SalI and SgfI with probe e and with probe c (orange bar). (B) Southern blot analysis of BamHI-digested ES-cell DNA of gene targeting with TV1 as outlined in A. A 6.2 kb fragment was observed in wild-type clones (+/+), and an additional 9.2 kb fragment in recombined clones (+/lacZ). (C) Southern blot analysis of BamHI-digested ES-cell DNA of second gene targeting (TV2). Besides a 12 kb wild-type fragment, a 7 kb fragment was detected in successfully targeted clones (+/hyg). (D) Pulse-field electrophoresis separation of SalI/SgfI-digested ES-cell DNA of double-targeted clones analyzed by Southern blot, hybridized with probe e (left) or probe c (right). Events on the same allele are easily distinguishable by the appearance of a 46 kb fragment in both panels (arrowhead) in addition to the wild-type fragment (arrow). In clones with trans orientation, an additional fragment of >150 kb is visible with probe e (left, white arrow) and a different fragment of 90 kb with probe c (right, white arrow).

View larger version (66K): [in a new window]   Fig. 2. E-cadherin-specific expression is lost in ES cells after deletion of intron 2. (A) Genotyping of Ecad-In2flox (+/flox) and Ecad-In2floxdel (+/del) ES cells after transient expression of Cre (left) and offspring of corresponding knock-in mouse strains (right). Primers specific for the floxed (upper panel) and floxdel allele (lower panel) were used. (B) Southern blot analysis of KpnI-fragmented DNA of Ecad-In2flox ES cell clones after Cre expression, using a radioactive probe specific for exon 2. The restriction fragments of the wild-type and Ecad-In2flox alleles migrate at 7.7 kb (black arrow), whereas that for the Ecad-In2floxdel allele migrates at 2.7 kb (white arrow). (C,D) X-gal staining of Ecad-In2flox (C) and Ecad-In2floxdel ES cells (D) shows that expression is lost upon deletion of intron 2 sequences. (E-H) Analysis of differentiated ES cells in teratomas. Expression of ß-gal is seen in teratomas from Ecad-In2flox cells (E), and this is significantly reduced in teratomas without intron 2 (F). (G,H) Sections of teratomas shown in E,F counterstained with Hematoxylin/Eosin. High-level expression in cystic epithelia in Ecad-In2flox (G) is lost in Ecad-In2floxdel teratomas. (I) Semi-quantitative (upper panel) and real-time PCR (lower panel) of both ES-cell lines with primers specific for betageo and Gapd transcripts. A reduction in gene activity is observed in the semi-quantitative and real-time PCR. Values resulting from Gapd real-time PCR were used for standardization. Scale bars: 50 µm in C,D,G,H; 500 µm in E,F.

View larger version (82K): [in a new window]   Fig. 3. Deletion of intron 2 leads to loss of ß-gal expression during early embryogenesis. (A-E) Whole-mount X-gal staining of Ecad-In2flox embryos between E6.5 and E10.5. (F,G) Paraffin sections of whole-mount stained Ecad-In2flox embryos. Transverse section at E7.5 (F) indicates expression in ectoderm and endoderm, but not in mesoderm. Expression levels appear higher in posterior ectoderm. Sagittal section at E10.5 (G) shows high-level ß-gal expression in pharynx and gut epithelium. Low-level expression in surface ectoderm is not visible in sections at this stage. (H-L) Whole-mount X-gal staining of Ecad-In2floxdel embryos between E6.5 and E10.5. Expression is only observed at low levels in AER and yolk sac at E10.5 (L). (M,N) Paraffin sections of whole-mount stained Ecad-In2floxdel embryos. No ß-gal expression is observed in sections of E7.5 embryos (M, transverse section) or inside of E10.5 embryos (N, sagittal section). (O) Semi-quantitative (upper panel) and real-time PCR (lower panel) of embryonic cups of Ecad-In2flox (+/flox) and Ecad-In2floxdel (+/del) embryos, similar to Fig. 2. A reduced signal is observed in +/del samples. For each PCR, a control without reverse transcriptase (-RT) is given. Transcript amounts were calculated from real-time PCR to be 85% reduced in Ecad-In2floxdel samples (lower panel). Scale bars: 100 µm in A,H; 250 µm in B,C,G,I,J,N; 500 µm in D,E,K,L; 50 µm in F,M.

View larger version (88K): [in a new window]   Fig. 4. Decreased expression of the E-cadherin reporter gene after deletion of intron 2 sequences at later embryonic stages. (A-C,F-H) Ecad-In2flox (A-C) and Ecad-In2floxdel embryos (F-H) were stained for ß-gal expression, at the indicated developmental stages, for 45 minutes (45', C,H) or overnight (ON, A,B,F,G). Increased expression in the skin is observed in Ecad-In2flox embryos during development. The E-cadherin locus without intron 2 is activated, but expression levels are significantly lower. (D,E,I,J) Isolated organs of E16.5 Ecad-In2flox (D,E) and Ecad-In2floxdel embryos (I,J) stained for ß-gal expression for 45 minutes (D,I) or overnight (E,J). High expression levels are found in the pancreas, stomach, gut and thymus of Ecad-In2flox embryos (D). After 45 minutes of staining, expression in organs of Ecad-In2floxdel embryos is only detected in pancreas and esophagus (I). After overnight incubation, lung epithelium is only weakly stained (J). (K-X) High magnification of sagittal sections of E11.5 embryos with the Ecad-In2flox (K-Q) and Ecad-In2floxdel allele (R-X). Organs or regions of the embryo are labeled in each figure. After sectioning, E-cadherin-specific expression can be observed in all tissues in Ecad-In2flox embryos, but no expression is found after deletion of intron 2, except for a faint expression detected in the pancreas primordium (V). Scale bars: 1 mm in A-J; 100 µm in K-X.

View larger version (67K): [in a new window]   Fig. 5. E-cadherin-specific expression in the yolk sac is independent of cis-regulatory elements in intron 2. (A-D) Whole-mount ß-gal staining of wild-type (A,C) or Ecad-In2flox (B,D, left) and Ecad-In2floxdel embryos and yolk sacs (B,D, right) at E10.5 (A,B) and E12.5 (C,D). Expression level in the yolk sac shows the same intensities in Ecad-In2flox and Ecad-In2floxdel embryos at all analyzed stages. (E) Semi-quantitative (upper panel) and real-time PCR (lower panel) of betageo (left) and Gapd transcripts (right) in yolk sacs at E10.5 and E16.5. No significant difference is observed in the level of expression between the two different strains. Scale bar: 1 mm.

View larger version (54K): [in a new window]   Fig. 6. Cells in the lens and salivary glands absolutely require cis-regulatory elements in intron 2 for activation of ß-gal transcription. (A-F) X-gal staining in lenses of Ecad-In2flox embryos shows high intensities (A,C,E), whereas no expression is found in lenses of Ecad-In2floxdel embryos (B,D,F). Lenses at stages E10.5 (A,B), E12.5 (C,D) and E14.5 (E,F) are shown. (G-P) Analysis of E-cadherin-specific expression in mandibular salivary (arrow) and thyroid (white arrowhead) glands at E16.5 (black arrowhead marks meninges). Heads of Ecad-In2flox (G) and Ecad-In2floxdel embryos (H) were cut prior to X-gal staining and viewed from bottom. In Ecad-In2flox high expression is found in the salivary and thyroid glands (G), but no staining is observed in salivary glands of Ecad-In2floxdel embryos (H). (I-P) Transverse sections of Ecad-In2flox (I-L) and Ecad-In2floxdel heads at E16.5 (M-P). Sections of salivary (I,M) and thyroid glands (J,N), trachea (K,O) and meninges (L,P). Scale bars: 100 µm in A,B,J,N; 250 µm in C,D; 500 µm in E,F; 1 mm in G,H; 50 µm in I,K,L,M,O,P.

View larger version (80K): [in a new window]   Fig. 7. Intron 2 sequences are required for maintaining E-cadherin expression. (A-D) Whole-mount ß-gal staining of F1 embryos of CK14-Cre males crossed to Ecad-In2flox females. A slightly reduced staining is seen at E12.5 (A) in embryos where Cre was active (+/) compared with control embryos with no Cre allele (+/flox). Further reduction is found in E13.5 (B), E14.5 (C) and E16.5 (D) embryos. Tissues where Cre was not active (lens, gut loops) are still strongly stained. (E,F) Whole-mount ß-gal staining of F1 embryos of CK19-Cre males crossed to Ecad-In2flox females. No difference in gene activity is observed at E9.5 (E), but decreased gene activity after intron 2 deletion is visible in E10.5 embryos (F). (G-J) Transverse sections of the gut tube (G,H) and sagittal sections of the pharynx (I,J) of whole-mount stained E10.5 control (+/flox, G,I) and Ecad-In2flox/CK19-Cre embryos (+/, H,J). Scale bars: 500 µm in E; 1 mm in A,B,F; 2 mm in C,D; 50 µm in G-J.

View larger version (13K): [in a new window]   Fig. 8. Model of E-cadherin regulation in the cadherin cluster. Parts of the P- and E-cadherin locus are shown. Exons are represented by vertical black bars and numbers, DHSs by vertical arrows, transcription start sites by small horizontal arrows, E-boxes by red boxes and sequences with enhancing activities by green boxes (intron 1-enhancer represented by unlabeled box). alt., sequences that mediate alternative, intron 2-independent gene activation in late embryogenesis; brain, sequences that contribute to brain-specific expression; endoderm, sequences required for endoderm-specific expression; enh., sequences that generally enhance transcription; sil., brain-specific silencer that restricts gene activity to E-cadherin expression domains; tse1-4, tissue-specific enhancers, including elements for ectoderm-specific expression. The presence of a locus control region (LCR) is not yet proven and the precise positions of `sil.', `tse1-4' and `alt.' are unknown. Elements that contribute to expression in yolk sac must be located outside of intron 2. E-boxes are required for downregulation of the gene (red arrow), whereas elements in intron 2 activate the locus (green arrows). The postulated LCR might influence gene activity for proper activation and downregulation (purple arrow).