DEV02647 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Structural comparison of the Chd-kielin family. (A) Domain structures of the Cv2, Kcp, kielin and Chd proteins. For mouse KCP, an alternative splicing product that lacks the sequence downstream of the arrow site has also been reported (Lin et al., 2005). (B) Amino acid sequence identities between Cv2 homologues. (C) A phylogenetic tree generated by ClustalW. The N-J tree with branch length program was used. The sequences are recorded in the database as follows: chick Cv2 (Accession Number NP_001007081), Drosophila Cv2 (Accession Number NP_524809), human Cv2 (Accession Number NP_597725), mouse chordin (Accession Number NP_034023), mouse Cv2 (Accession Number NP_082748), mouse Kcp (Accession Number BAE32541), Sog (Accession Number NP_476736), Xenopus Cv2 (Accession Number AAX12852), xtCv2 (Accession Number AAX12853), Xenopus Kielin (accession number BAA95483) and zebrafish Cv2 (Accession Number AAX14720). CR, chordin-type cystein-rich repeat; SP, signal peptide; TIL, trypsin inhibitor-like domain; vWD, von Willebrand factor D domain.

• Supplemental Figure 2 -

  Fig. S2. Generation of the second mouse Cv2 knockout line with an nlacZ-knock-in vector, and of the Kcp knockout line. (A) Construction of the targeting vector for an nLacZ allele of mouse Cv2. In the mutant allele, the first codon of mouse Cv2 was replaced in-frame with the first methionine of β-galactosidase with a nuclear localizing signal (nLacZ). The locations of the PCR primers used for recombinant ES cell selection (black arrows) and genotyping (open arrowheads), and the probes for Southern blot analysis are indicated (black boxes). Bg, BglII; Sc, SacI. (B) Construction of the targeting vector for Kcp knockout mice. In the mutant allele, the first codon of Kcp was replaced in-frame with the first methionine of the tau-lacZ sequence. The locations of the PCR primers used for recombinant ES selection (blue arrows) and genotyping (open arrowheads), and probes for Southern blot are indicated (orange and black boxes). Bg, BglII; HIII, HindIII; RI, EcoRI. (C,D) PCR selection (C) and Southern blot analysis (D) for the Kcp recombinant ES cells. (E) PCR genotyping of each Kcp allele in mutant mice. Genomic DNA was prepared from the tails of wild-type, heterozygous and homozygous mice (left to right). Allele-specific PCR fragment bands were detected at 553 bp for the wild-type allele and 390 bp for the mutant allele.

• Supplemental Figure 3 -

  Fig. S3. RT-PCR analysis. Trunk tissues of the control (left) and Cv2^−/− (right) mice on E14.5. The expression levels of aggrecan 1 and Col2a1 were reduced in the Cv2 mutant (bracket). Those of Pax1, Sox9 and Bmp genes were largely unaffected.

• Supplemental Figure 4 -

  Fig. S4. Impaired lung maturation in Cv2^−/− mice. (A-D) Histological analysis on P0. (B,D) The alveoli were not fully inflated in the null mouse Cv2 mutant. (C,D) Higher magnification views. (E,F) On E18.5, no size difference was observed between the control (Cv2^+/+, Cv2^+/−) and Cv2^−/− lungs. (G,H) Proper expansion of the lung tissues in the peripheral region (translucency) was not observed in the Cv2^−/− mutant. (I-L) Histological analysis revealed that expansion of the alveoli was impaired in the mutant lung (J,L). (K,L) Higher-magnification views. (M-V) Immunostaining of cell-type-specific markers in the lung. SP-C, a marker for alveolar type II cells of the distal epithelium; AQP5, a marker for alveolar type I cells of the distal epithelium; aSMA, a marker for smooth muscle cells surrounding the blood vessels and proximal bronchioles; CC10, a marker for Clara epithelial cells of the proximal epithelium; PECAM, a marker for blood vessels. Expression of these markers was largely unaffected in the Cv2^−/− mutant (N,P,R,T,V), except that the SMA staining was found in more-peripheral regions of the lung (i.e. closer to the pleura) in the mutant than in the control. Genotypes are indicated in the lower left corner of each panel.

• Supplemental Figure 5 -

  Fig. S5. Dorsoventral polarity in the neural tube was largely unaffected in the Cv2^−/− mutant mouse embryo. (First six rows) Immunostaining was performed using the region-specific markers for the neural tube with the control (left) and Cv2^−/− mutant (right) embryos at E10.5. The shape of the neural tube is indicated with broken lines. (Bottom row) HE staining of cross-sections of the E12.5 embryos. The neural tube regions of the control (left) and Cv2^−/− mutant (right) embryos are shown.