DEV01662 Supplemantary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Segment of the VISTA plot of evolutionary DNA sequence conservation of the genomic region of the E-cadherin locus in mouse, human, chimp, rat and dog. The following sequences containing Cdh1 were used: mouse, Chr. 8 107800000- 108300000; human, Chr. 16 68100000-68900000; chimp, Chr. 18 61300000-62000000; rat, Chr. 19 36150000-36700000; dog, Chr. 5 83600000-84300000. Comparative analyses were performed using VISTA (http://www-gsd.lbl.gov/vista/index.shtml). Percent nucleotide identities between sequences of each species and mouse are plotted as a function of positions along the mouse sequence. The genes for P- and E-cadherin are labeled with gray arrows marking a region between –10 kb upstream of the transcription start site and the last exon. The light gray bar between 316 and 349 kb marks the region of previous DNaseI-hypersensitive site (DHS) mapping. Positions of DHSs are shown by arrows and as yellow shading across all plots. E-cadherin intron 2 is located between 332.5 and 377 kb. Peaks of evolutionary conservation for each alignment are shaded blue for exons and pink for aligned non-coding regions to show cases of more than 70% identity over 100 bp. Significant evolutionarily conserved regions in E-cadherin intron 2 located outside of thus far mapped positions of DHSs are additionally labeled by green shading. The chimp sequence is incomplete, having many gaps of unknown sequence, which made it impossible to detect all peaks of evolutionary conservation. In the region 5˘ to the transcription start site of E-cadherin (331.2 kb) significant conservation between mouse, human, chimp, rat and dog is present only at the promoter, at approx. –1.2 kb (329.8 kb), –2.3 kb (328.9 kb) and –3.4 kb (327.8 kb), regions that were all included in previous transgene analysis for cis-regulatory sequences (Stemmler et al., 2003). Going upstream up to –10 kb (321 kb), no additional conserved area is found, and only four peaks are present further upstream as far as the 3˘ end of P-cadherin (313.2, 307.2, 292.3 and 287.5 kb). Only one peak of conservation is detected 3˘ of exon 16 of E-cadherin (405 kb), going as far as to the adjacent gene coding for an Armadillo-repeat protein (398-410 kb). Several blocks with evolutionary conservation appeared in intron 2. The mapped DNaseI-hypersensitive sites (DHSs) colocalized with these areas (yellow shading and arrows). Only two DHSs (VI and ‘*’) shared no significant identity in all analyzed species. In addition to the DHS-mapped region that has been analyzed in transgenic mice (Stemmler et al., 2003), several peaks with a high degree of sequence conservation are distributed throughout intron 2 (green shaded areas, Fig. S1). This shows that positions of DHSs correlate with regions of evolutionary sequence conservation and further indicates the presence of cis-regulatory elements at these positions.

• Supplemental Figure S2 -

  Fig. S2. E-cadherin-betageo reporter knock-in mice faithfully recapitulate E-cadherin expression patterns. To verify whether the introduced reporter gene is able to recapitulate the endogenous E-cadherin expression, a variant of the targeting vector TV1 (Fig. 1A), lacking the loxP site, was used to generate a mouse strain with a targeted allele (Ecad-ATG), and the betageo expression pattern was compared to the E-cadherin mRNA distribution during embryonic development. (A-H) E-cadherin in situ hybridization of whole-mount embryos (A-C) and on paraffin sections (D˘,E˘,F˘,G˘,H) of indicated stages between E6.5 and E16.5. Adjacent sections were histologically stained with Hematoxylin-Eosin (D-G). E-cadherin transcripts at late gastrulation stages are found throughout the extra-embryonic and embryonic tissues (A). On sections at this stage (E7.5), E-cadherin mRNA is predominantly present in the ectoplacental cone and the primitive ectoderm as well as in the visceral endoderm, but is rare or absent in the visceral yolk sac and in the mesoderm (E˘). At early gastrulation (E6.5), the expression levels are high in the extra-embryonic ectoderm, but weaker in the epiblast (D˘). Later, at E8.5, E-cadherin mRNA is found in the gut tube (definitive endoderm) and at slightly reduced levels in the surface ectoderm (B,F˘,G˘), but not in the neurectoderm (F˘). At E11.5, the highest expression in the ectoderm is observed in the facial region of the mandibular components of the first branchial arch, in the AER (C) and in the lens (not shown). In addition, from E8.5 onwards, expression is found in the yolk sac (B). During organogenesis, E-cadherin mRNA is detected in all epithelia of the lung, kidney, gut, stomach and skin, as well as in the liver (H). (I-T) Whole-mount X-gal staining of Ecad-ATG embryos of the indicated stages reflects E-cadherin-specific expression patterns as found by in situ hybridization. b-Gal expression is in addition observed in the ventral midbrain, shown at higher magnification in O (arrow), consistent with the observations of Shimamura et al. (Shimamura et al., 1994). At E14.5 all epithelia in inner organs show high b-gal activity (S-T). (U-V) Sagittal (U,W,Y) and transverse sections (V,X) of whole-mount stained embryos, counterstained with Eosin. The reporter gene activity parallels the expression pattern of E-cadherin in early embryogenesis and organogenesis, and it accurately reflects down-regulation in the mesoderm at gastrulation and in neurectoderm at neurulation. Hence, the betageo knock-in allele allows us to correctly monitor spatiotemporal alterations in E-cadherin gene activity. Plane of sections in V,X is shown in U,W, respectively. Orientation of embryos: anterior towards the left (A,B,E-F’,K,U-X) or dorsal towards the right (C,G-H,L-R,Y). al, allantois; am, amnion; bl, bladder; dc, decidua; ea, ear; ec, ectoplacental cone; ect, ectoderm; eex, embryonic-extraembryonic border; end, endoderm; es, esophagus; fg, foregut diverticulum; gl, gut loop; hf, head fold; hg, hindgut diverticulum; hn, Hensen’s node; ht, heart; kd, kidney; l, lens; lu, lung; lv liver; mes, mesoderm; nc, nasal cavity; ne, neurectoderm; op, optic eminence; ot, otic pit; pa, pancreas; ph, primordia of hair follicle; ps, primitive streak; pv, primordia of vibrissae; rm, Reichert’s membrane; se, surface ectoderm; sk, skin; so, somites; st, stomach; th, thymus; tr, trophectoderm; tra, trachea; ur, ureter; vy, visceral yolk sac. Scale bars: 250 mm in A,B,F-G˘,K-N; 1 mm in C,O-T,Y; 100 mm in D-E˘,J,U-X; 2 mm in H; 50 mm in I.