DEV015495 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Schematics of the wild-type and four known targeted alleles of Wnt7b. Exon-intron boundaries are not to scale. Bridging diagonal black lines indicate the splicing of exons. Exon 1a is shown with its initiation codon; it also contains a signal sequence. The alternative exon 1, labeled exon 1b, is also shown with its own initiation codon. Exon 1b similarly contains a highly conserved peptide signal sequence. Wnt7b⁺ is the wild-type allele. Wnt7b^D3-4 is the original null allele of Parr et al. (Parr et al., 2001) in which most of exon 3 and all of exon 4 are replaced with a neomycin resistance cassette. Wnt7b^lacZ is the hypomorphic allele of Shu et al. (Shu et al., 2002) in which exon 1a is replaced by a lacZ insertion and a neomycin resistance cassette with its own promoter. This allele allows alternative splicing through exon 1b. The Wnt7b^D1 allele is the hypomorphic allele of Lobov et al. (Lobov et al., 2005) in which Cre-mediated recombination has removed exon 1a, which encourages alternative splicing through exon 1b. Finally, Wnt7b^D3 is the true null allele that we present in this paper, in which exon 3 is removed by Cre-mediated recombination. This allele leaves no extra genetic elements in the Wnt7b locus, and is insensitive to the alternative splicing of exon 1.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Wnt7b consists of two isoforms that utilize alternative first exons. (A) A ³²P-labeled RNA probe that spans exons 1 and 2 of Wnt7b demonstrates the presence of two isoforms of Wnt7b: a larger protected fragment that is of the predicted size of the intact protected probe (top arrow) and a second, smaller fragment that is protected by exon 2 only (bottom arrow). Genotypes are indicated. (B) RNase protection was performed using a ³²P-UTP probe generated from a 314 bp fragment spanning the exon 1/2 junction which was amplified by PCR using the following primers: Wnt7b RPA for 5′-GGCCCGCAGCCCGGCGC-3′ and Wnt7b Rpa rev 5′-GTTCTTGCCCGAAGCGGTC-3′. This probe was hybridized to 10 µg of total mRNA from E14.5 lung, digested with RNase according to the procedures outlined in the RPA II Kit (Ambion, #AM1410) and then run on a 6% denaturing polyacrylamide gel. β-actin was used as a control according to the manufacturer’s recommendations. (C) 5′ RACE identifies a second isoform of Wnt7b that possesses an alternative first exon that we termed Wnt7b-2 (red) to distinguish it from the previously predicted isoform Wnt7b-1 (green). Translation of the open reading frame of Wnt7b-2 (red) revealed a start methionine that was upstream of and in frame with exon 2 of Wnt7b. The alternative exon 1b also codes for a highly conserved peptide signal sequence. 1 µg of total RNA was extracted from E14.5 wild-type and Wnt7b^D3-4/D1 lungs with Trizol (Invitrogen). cDNA synthesis and 5′ RACE were carried out using a 5′ RACE System Kit, version 2.0 (Invitrogen, #18374-058). Two gene-specific primers were used: Gsp1, 5′-GTCGGCTCTGGCAAGATGGC-3′ and Gsp2, 5′-CAGGCCAGGAATCTTGTTGC-3′. A PCR fragment of approximately 600 bp was cloned and sequenced. For analysis of potential signal peptides, the open reading frame was analyzed using SigFind and SigX software. (D) RT-PCR revealed the presence of both isoforms of Wnt7b in E9.5 whole embryos and lung (lanes 1-4). The relative abundance of Wnt7b-2 increases in embryos in which exon 1a has been deleted (compare lanes 2 and 4). No product is detected in control samples lacking reverse transcriptase (lanes 5 and 6). RT-PCR was performed using a single reverse primer in exon 2 (5′-TCCCTACTCGGAGTTCTTGC-3′) and a primer specific to exon 1A (5′-acgtgtttctctgctttggc-3′) or exon 1B (5′-CTTTCTCCTTCTGTCCAGCG-3′). The expected sizes of the fragments were 270 and 260 bp for the exon 1A and 1B products, respectively. The 270 bp fragments reflecting Wnt7b-1 (which begins with exon 1A) appear in lanes 1, 3 and 5, whereas the presumptive 260 bp fragments amplifying Wnt7b-2 (beginning with exon 1 Bb) have been run out in lanes 2, 4 and 6. Genotypes of animals are indicated.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Complete recombination results in a loss of Wnt7b mRNA throughout the embryo. (A) ISH shows complete loss of Wnt7b mRNA in E14.5 lung endoderm. Arrowheads point to the epithelial boundary demarcated by dotted lines. (B) RT-PCR data from littermates of the cross depicted in Fig. 1A. Genotypes are indicated. RT-PCR shows complete recombination of the conditional Wnt7b^C3 allele, thereby converting Wnt7b^C3 into the null Wnt7b^D3 allele. This occurs faithfully whenever Sox2Cre is present in the embryo. RNA was harvested from the entire E9.5 embryo demonstrating that recombination occurred throughout all embryonic tissues. Wnt7b transcripts were amplified by PCR with Wnt7b exon 2 forward primer (5′-GTCTTCGGGCAAGAACTCC-3′) and Wnt7b exon 4 reverse primer (5′-ACCGCTGCGTTGTACTTCTC-3′), which generates a 434 bp fragment from both the wild-type and the unrecombined Wnt7b^C3 allele (C3). These same primers generate a 193 bp fragment from the recombined Wnt7b^D3 (D3) allele. Control RT-PCRs without reverse transcriptase (-RT) are shown.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Deletion of Wnt7b does not cause vascular smooth muscle abnormalities. (A-B′) Hematoxylin and Eosin staining of paraffin lung sections from control and mutant P0 pups. Hemorrhage (solid arrowheads) is visible around large, proximal blood vessels (A′) and in distal alveoli (B′) in mutant lungs. No hemorrhage is seen in controls (A,B). Thickness of vasculature smooth muscle is unchanged in the mutant (open arrowheads). (C-D′′) Immunohistochemistry of paraffin lung sections from control and mutant P0 pups. Blood vessels and surrounding smooth muscle, as marked by PECAM (red) and smooth muscle actin (green), respectively (C,C′), appear normal in vessels adjacent to large airways. Smooth muscle actin (red) and TUNEL (green) double staining reveals no apoptosis in smooth muscle surrounding large blood vessels (D,D′). Rare TUNEL-positive cells in adjacent airways are circled. Paraffin sections of irradiated guts serve as a positive control for the TUNEL stain (D′′).

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. Expression of many factors affecting lung growth is preserved, whereas expression of Bmp4, Id2 and, to a lesser extent, Fgf10, is reduced. (A-F′,H-J′,L-P′) ISH of mutant and control E14.5 lungs. (G-G′,K-K′) ISH of mutant and control E12.5 lungs. Epas1 expression is unchanged in mutant lungs (A,A′). Id2 expression is reduced in mutant lungs compared with control (B,B′). Expression of Nkx2.1 (C,C′), Sox9 (D,D′) and Tgfb1i1 (E,E′) is unchanged in mutant lungs. Epithelial Bmp4 expression is reduced in mutant lungs compared with control (F,F′). Expression of Gli1 (G,G′), Hip1 (H,H′), Ptch1 (I,I′) and Shh (J,J′) is unchanged in mutant lungs. Fgf10 expression is variably reduced in the distal interbud mesenchyme in mutant compared with control lungs at E12.5 and E14.5 (K,K′,L,L′). Epithelial and mesothelial Fgf9 expression is unchanged in mutant lungs (M,M′). A segment of mesothelium is removed from control lungs to demonstrate superficial mesothelial and deeper epithelial staining (indicated by the open arrowhead). Expression of Wnt2 (N,N′), Wnt5a (O,O′) and Wnt11 (P,P′) is unchanged in mutant lungs.