Mice exclusively expressing the short isoform of Smad2 develop normally and are viable and fertile

Abstract

Smad2 and Smad3 are closely related effectors of TGFbeta/Nodal/Activin-related signaling. Smad3 mutant mice develop normally, whereas Smad2 plays an essential role in patterning the embryonic axis and specification of definitive endoderm. Alternative splicing of Smad2 exon 3 gives rise to two distinct protein isoforms. The short Smad2(Deltaexon3) isoform, unlike full-length Smad2, Smad2(FL), retains DNA-binding activity. Here, we show that Smad2(FL) and Smad2(Deltaexon3) are coexpressed throughout mouse development. Directed expression of either Smad2(Deltaexon3) or Smad3, but not Smad2(FL), restores the ability of Smad2-deficient embryonic stem (ES) cells to contribute descendants to the definitive endoderm in wild-type host embryos. Mice engineered to exclusively express Smad2(Deltaexon3) correctly specify the anterior-posterior axis and definitive endoderm, and are viable and fertile. Moreover, introducing a human Smad3 cDNA into the mouse Smad2 locus similarly rescues anterior-posterior patterning and definitive endoderm formation and results in adult viability. Collectively, these results demonstrate that the short Smad2(Deltaexon3) isoform or Smad3, but not full-length Smad2, activates all essential target genes downstream of TGFbeta-related ligands, including those regulated by Nodal.

Figure 1.

Smad2 and Smad3 structural comparisons. (A) Mouse Smad2 and Smad3 genomic organization. The alternatively spliced Smad2 exon 3 and the corresponding 30-amino acid insert it encodes are shown in red. Noncoding exons are depicted as open rectangles. Smad2 exons 4–11 and Smad3 exons 2–9 are nearly identical in size. (B) Smad2 and Smad3 protein alignments showing the highly conserved N-terminal and C-terminal Mad Homology domains 1 (MH1) and 2 (MH2), respectively, and the intervening proline-rich linker region. The Smad2 MH1 domain also contains an N-terminal glycine-rich 10-amino acid insert (yellow) that is not present in Smad3. Smad2 and Smad3 possess distinct C-terminal SS(V/M)S phosphorylation motifs. The sequences corresponding to those protected by RPA probes are indicated as black lines.

Figure 2.

Smad2 splice variants are coexpressed during early mouse embryonic development. (A) Quantitative analysis of Smad2(FL) and Smad2(Δexon3) expression levels in CCE ES cell and embryonic total RNA at various stages. The size of the fragments protected by RPA probe A (Fig. 1B) is indicated. The ratio of Smad2(FL) to Smad2(Δexon3) expression decreases with developmental age. The expression ratios indicated by the numbers at the bottoms of the lanes were calculated by scanning the gels in a PhosphorImager and measuring the amount of radioactivity in each band to determine the precise ratios. (B) Total Smad2 transcript levels monitored with RPA probe B (Fig. 1B).

Figure 4.

Production of mutant mice exclusively expressing the alternatively spliced Smad2Δexon3 isoform. (A) Strategy used to replace exon 3 with a loxP (blue triangles)-flanked neomycin-resistance cassette. Targeted clones were identified with a 5′ external probe (black line). (E) EcoRI; (H) HindIII; (B) BamHI; (X) XbaI; (K) KpnI; (Nc) NcoI. (B) Southern blot analysis. The 5′ external probe distinguishes 6.8-kb wild-type (+/+) and 5.1-kb targeted (Δex3/+) alleles. (C) PCR genotyping of wild-type, Smad2^Δexon3/+ (Δex3/+), and Smad2^{Δexon3/Δexon3} (Δex3/Δex3) adult mice. The PCR primers in A give rise to products at the indicated sizes. (D) Total RNA from adult tissues was analyzed with RPA probe A (Fig. 1B), which distinguishes Smad2(FL) and Smad2(Δexon3) transcripts. Smad2 (Δexon3) mRNA expression is up-regulated in Smad2^Δexon3/+ mice, whereas Smad2(FL) transcripts are undetectable in Smad2^Δexon3 homozygous mutants. (E) Endogenous Smad2 (probe B) and Smad3 (probe C) expression levels are unperturbed in Smad2^Δexon3 homozygous mice. The expression ratios indicated by the numbers at the bottoms of the lanes were calculated by scanning the gels in a PhosphorImager and measuring the amount of radioactivity in each band to determine the precise ratios. (F) Western blot analysis using anti-phospho-Smad2 (α-PSmad2) reveals robust Smad2(FL) phosphorylation in wild-type B and T lymphocytes and HepG2 hepatocytes stimulated with TGFβ1, whereas Smad2(Δexon3) is efficiently phosphorylated in Smad2^Δexon3 homozygous mice (lanes 6,12). (sp) Spleen; (th) thymus; (k) kidney; (lu) lung; (li) liver.

Figure 3.

Reconstituted Smad2-deficient ES cells expressing Smad2(Δexon3) or Smad3, but not Smad2(FL), contribute to the definitive endoderm in chimeric embryos. (A) Vector design and electroporation scheme. pCAGGS contains a chimeric promoter between the cytomegalovirus (CMV) immediate-early enhancer and the chicken β-actin promoter as well as the chicken β-globin intron and rabbit β-globin polyadenylation signals. cDNAs encoding N-terminally Flag-tagged human (h) Smad2(FL), Smad2(Δexon3), or Smad3 were introduced into Smad2^Robm1 homozygous ES cells. Hygromycin-resistant clones were initially screened by flow cytometry for production of a human Smad2/3 protein. Smad2/3 expression levels in selected clones were subsequently analyzed via flow cytometry in experiments done synchronously with microinjection assays to verify that transfected ES cell clones tested in chimeras express roughly equivalent amounts of protein. A representative FACS is shown. (B) Western blot analysis reveals efficient production of Flag-hSmad2(FL), Flag-hSmad2(Δexon3), and Flag-hSmad3. The background band that comigrates with Flag-hSmad3 is detectable using the M5 monoclonal alone. (FL) Flag-hSmad2(FL); (Δex3) Flag-hSmad2(Δexon3); (S3) Flag-hSmad3. (C) Contribution to the definitive germ layers in E9.5 chimeric embryos. Transverse sections of representative chimeras stained for β-galactosidase activity at foregut (asterisk), midgut (mg), and hindgut (hg) levels. Wild-type R26.1 as well as KT11-Smad2(Δexon3)- and KT15-Smad3-expressing ES cells robustly colonize host embryos and give rise to descendants in all embryonic lineages, including the definitive endoderm. In contrast, Smad2-deficient KT15- and KT11-Smad2(FL)-expressing ES cells fail to contribute to the definitive endoderm, but efficiently form mesoderm and ectoderm.

Figure 5.

Replacement of endogenous Smad2 coding sequences with Smad3. (A) The ATG-containing (arrow) exon 2 of Smad2 was replaced with an N-terminally Flag-tagged human (h) Smad3 cDNA followed by a polyadenylation signal and loxP (blue triangle)-flanked neomycin-resistance cassette. Targeted clones were identified with a 5′ external probe (black line). (B) BamHI; (E) EcoRI; (H) HindIII. (B) Southern blot analysis. The 5′ external probe detects 11.5-kb wild-type (+/+) and 5.1-kb targeted (3ki/+) alleles. (C) PCR analysis of DNA samples from wild-type, Smad2^hSmad3ki/+ (3ki/+), and Smad2^{hSmad3ki/hSmad3ki} (3ki/3ki) adult mice. PCR primer locations are indicated in A. (D) Expression of the introduced hSmad3 cDNA and endogenous Smad2 by RPA of ES cell, human HepG2 hepatoma, adult tissues, and E12.5 total RNA. (Lanes 3,4) A 3′ human Smad3 RPA probe fails to protect mouse mRNA sequences. Similarly, probe B (Fig. 1B) is specific for mouse Smad2 (cf. lanes 2 and 1,3–8). The hSmad3 RPA probe reveals a polymorphism between the HepG2 Smad3 cDNA and the human Smad3 cDNA used in our studies (cf. lanes 1 and 2). (Lanes 1,5–8) Small differences in the ratio of human Smad3 expression from the Smad2^hSmad3ki allele to Smad2 expression from the wild-type allele are observed in Smad2^hSmad3ki/+ ES cells and mice. (Lanes 9–11) Transcription from the remaining downstream exons in the Smad2^hSmad3ki targeted locus is nearly undetectable. The expression ratios indicated by the numbers at the bottoms of the lanes were calculated by scanning the gels in a PhosphorImager and measuring the amount of radioactivity in each band to determine the precise ratios. (E) Whole-mount views of E12.5 Smad2^hSmad3ki homozygous embryos (3ki/3ki) derived from a homozygous parental intercross. Embryos are ordered from overt wild-type morphology (left) to increasingly more severe anterior abnormalities (right).