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. 2004 Jun 15;18(12):1482-94.
doi: 10.1101/gad.1202604.

In vivo convergence of BMP and MAPK signaling pathways: impact of differential Smad1 phosphorylation on development and homeostasis

Josée Aubin  1 , Alice Davy, Philippe Soriano
Affiliations

In vivo convergence of BMP and MAPK signaling pathways: impact of differential Smad1 phosphorylation on development and homeostasis

Josée Aubin et al. Genes Dev. .

Abstract

Integration of diverse signaling pathways is essential in development and homeostasis for cells to interpret context-dependent cues. BMP and MAPK signaling converge on Smads, resulting in differential phosphorylation. To understand the physiological significance of this observation, we have generated Smad1 mutant mice carrying mutations that prevent phosphorylation of either the C-terminal motif required for BMP downstream transcriptional activation (Smad1(C) mutation) or of the MAPK motifs in the linker region (Smad1(L) mutation). Smad1(C/C) mutants recapitulate many Smad1(-/-) phenotypes, including defective allantois formation and the lack of primordial germ cells (PGC), but also show phenotypes that are both more severe (head and branchial arches) and less severe (allantois growth) than the null. Smad1(L/L) mutants survive embryogenesis but exhibit defects in gastric epithelial homeostasis correlated with changes in cell contacts, actin cytoskeleton remodeling, and nuclear beta-catenin accumulation. In addition, formation of PGCs is impaired in Smad1(L/L) mutants, but restored by allelic complementation in Smad1(C/L) compound mutants. These results underscore the need to tightly balance BMP and MAPK signaling pathways through Smad1.

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Figures

Figure 1.
Figure 1.
Targeting of the Smad1 locus. (A) Introduction of mutations in the C-terminal end of the Smad1 gene encoded by exon 7 using homologous recombination. The PGKneo selection cassette flanked by LoxP sites (arrowheads) and the diphtheria toxin (DTA) cassette in the targeting construct are indicated. (B) Southern blot analysis of DNA from ES cells using BamHI digestion hybridized with a 5′-flanking probe, to distinguish between the mutant (2.7 kb) and the wild-type (10 kb) alleles. (C) Western blot analysis of protein extracts from wild-type and Smad1C mutant embryos showed that similar levels of Smad1 protein were detected for each genotype. RasGAP served as a loading control. (D) Introduction of the linker mutation by homologous recombination substituting a mutated exon 3. (E) Southern analysis using XbaI digestion and a 3′-flanking probe, to distinguish between the mutant (10 kb) and the wild-type (12 kb) alleles. (F) Western blot analysis of nuclear extracts from wild-type and Smad1L mutant MEFs showed that similar level of Smad1 protein was detected for each genotype. γ-Tubulin was used as a loading control. (B) BamHI; (N) NheI; (X) XbaI; (Xh) XhoI.
Figure 2.
Figure 2.
Smad1C mutant phenotype. Comparison of E9.5 wild-type (A) and Smad1C/C (B,C) embryos showed that the C-terminal mutation caused heart defects, abnormal turning, and lack of ventral closure in the posterior region. Compared with the Smad1-/- (D), the SmadC/C (B,C) mutants also displayed head (open white arrow) and branchial arch (open black arrow) anomalies, showed in insets. (E–J) Whole-mount in situ hybridization analyses showed stronger Eomes expression in extraembryonic tissues of Smad1-/- (F) and Smad1C/C (G) embryos at E7.5, compared with controls (E). Bmp4 expression was also abnormal in Smad1-/- (I) and Smad1C/C (J) mutants compared with the controls (H). The placental connection (K–N) was also defective in the Smad1C mutants (M,N). In wild-type (K,L), the allantois fused to the placenta (K,L). In contrast, growth of the allantois was often observed (M) but either not properly fused in the Smad1C mutants (N) or fused ectopically (data not shown). (a) Allantois; (p) placenta; (t) tail.
Figure 3.
Figure 3.
Stomach anomalies in Smad1L mutants. Adult stomachs of wild-type (A,D,G,J), Smad1L/L (B,E,H,K), and Smad1L/-; Meox2Cre/+ mutants (C,F,I,L) were analyzed by histological (A–C) and IHC staining (D–L). HE staining revealed a decrease in the zymogenic cells (brackets), and an increase in parietal cells in Smad1L mutants (B,C), compared with wild-type (A). (A–C, insets) Higher magnification of the zymogenic zone where parietal cells were abundant and zymogenic cells depleted in Smad1L/L and in Smad1L/-; Meox2Cre/+ mutants. The latter carry an Smad1L allele in the context of the mosaic deletion of the null allele by Meox2Cre/+-driven recombination. The increase in the number of parietal cells was clearly visible using an anti-H+K+ proton pump antibody specific for this cell type when comparing wild-type (D) with Smad1L/L and Smad1L/-; Meox2Cre/+ mutants (E,F). The latter carry a Smad1L allele in the context of the mosaic deletion of the null allele by Meox2Cre/+-driven recombination. (C) Furthermore, the gastric epithelium of Smad1L/-; Meox2Cre/+ mutants showed signs of disorganization. (G–I) Immunostaining using a β-catenin antibody revealed that parietal cells, recognizable by their “friedegg” shape, showed increased proportion of nuclear staining (white arrowhead; H,I), compared with wild-type (black arrowhead; G). (J–L) Changes in the localization of Smad1 protein were also observed in mutants. In wild-type (J), Smad1 was distributed throughout the majority of parietal cells (black arrow), whereas a strong cytoplasmic membrane and nuclear staining were found in mutants (white arrow; K,L). Furthermore, a small proportion of Smad1L/- mosaic mutants died at birth from a ruptured stomach (open arrowhead; M–O). (N,O) Histology showed thinning of both the epithelial and the muscular layer and the absence of cardia at the junction of the esophagus and the stomach. (d) Duodenum; (e) esophagus; (ep) epithelium; (m) muscular layer; (s) stomach; (sp) spleen.
Figure 4.
Figure 4.
Characterization of Smad1 mutant MEFs. Immunofluorescence analyses showed redistribution of Smad1 protein in Smad1L/L MEFs (B) compared with wild-type (A). Staining at the membrane was stronger in the former. β-Catenin staining indicated that Smad1L/L cells lose adhesion zippers (D) normally observed in wild-type cells (circle; C) and exhibit increased cadherin staining at the membrane (E,F). Relocalization of actin to the cortex and reduction in stress fibers were predominant in Smad1L/L MEFs (H), in contrast to wild-type cells (G). In wild-type MEFs stimulated with PDGF, treatment with the MAPK inhibitor U0126 (J) caused retention of the Smad1 protein at the membrane. This pattern was not observed in control conditions (I; data not shown).
Figure 5.
Figure 5.
Reproductive defects in Smad1L/L mutants. Gross morphology of the male reproductive tract (A,C) revealed smaller testes and abnormal epididymis in Smad1L/L males (C) compared with wild-type (A). Upon histology, the testis cord was disorganized in affected Smad1L/L mutants compared with controls (data not shown). Furthermore, whereas PGCs were readily detectable at the periphery of wild-type testis cord (yellow arrow; B), they were severely depleted in affected Smad1L/L males (black arrow; D). Sertoli cells were unaffected. (E–J) AP staining on gonad sections of E13.5 embryos showed that PGCs were strongly labeled (white arrow) in wild-type (E,F) and in a proportion of Smad1L/L mutants (G,H), whereas other Smad1L/L gonads were devoid of germ cells (red arrow; I,J). (e) Epididymis; (g) gonad; (s) stomach; (t) testis.
Figure 6.
Figure 6.
PGC formation at E7.5 in wild-type, Smad1L, and Smad1C mutants. E7.5 embryos were stained for AP (A–D) to detect PGC at the posterior side of the wild-type (A), Smad1L/L (B), Smad1C/C (C), and Smad1C/L (D) embryos. Compared with the number of PGCs observed in wild-type embryos (black arrow, A), some Smad1L/L mutants (B) and almost all Smad1C/C embryos (C) were depleted of PGCs (open arrow). PGC formation was restored in Smad1C/L mutants (black arrow; D). (E) The modal distribution of embryos according to their genotype and the PGC count is shown. Embryos were classified in three categories depending on the number of PGCs.
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