Activin type IIA and IIB receptors mediate Gdf11 signaling in axial vertebral patterning

Abstract

Vertebral bodies are segmented along the anteroposterior (AP) body axis, and the segmental identity of the vertebrae is determined by the unique expression pattern of multiple Hox genes. Recent studies have demonstrated that a transforming growth factor beta (TGF-beta) family protein, Gdf11 (growth and differentiation factor 11), and the activin type II receptor, ActRIIB, are involved in controlling the spatiotemporal expression of multiple Hox genes along the AP axis, and that the disruption of each of these genes causes anterior transformation of the vertebrae. Skeletal defects are more severe in Gdf11-null mice than in ActRIIB-null mice, however, leaving it uncertain whether Gdf11 signals via ActRIIB. Here we demonstrate using genetic and biochemical studies that ActRIIB and its subfamily receptor, ActRIIA, cooperatively mediate the Gdf11 signal in patterning the axial vertebrae, and that Gdf11 binds to both ActRIIA and ActRIIB, and induces phosphorylation of Smad2. In addition, we also show that these two receptors can functionally compensate for one another to mediate signaling of another TGF-beta ligand, nodal, during left-right patterning and the development of anterior head structure.

Figure 1

Axial vertebral patterning in activin receptor mutant mice. Representative vertebral patterning in wild-type (A,D,G,J), IIB^−/− (B,E,H,K), and IIA^+/− IIB^−/− (C,F,I,L) mice. (A–C) Ventral view of vertebral skeletons. Wild-type skeleton consists of 13 thoracic (T) and 6 lumbar (L) vertebrae. The skeleton pattern is altered to T16L6 in IIB^−/− and T17L7 in IIA^+/− IIB^−/− mice. (D–F) Increased number of vertebrosternal (VS) ribs in IIB^−/− and IIA^+/− IIB^−/− mice. Arrows in F indicate T1 and T2 ribs fused ventrally to VS2 rib. (G–I) Cervical and thoracic vertebrae showing transformation of C7 vertebra in IIA^+/− IIB^−/− mice. Tuberculi anterior (TA) is present at C6 in wild-type and IIB^−/− mice, whereas it is present at C7 in IIA^+/− IIB^−/− mice. Attached ribs indicate thoracic vertebrae. (J–L) Morphology of C6-T1 vertebrae showing C7 to C6 transformation in IIA^+/− IIB^−/− mice. Note that the tuberculi anterior is missing in C6 (asterisk) and the transverse foramen (TF) is present in C7 of IIA^+/− IIB^−/− mice.

Figure 2

Multiple developmental defects in IIA^+/− IIB^−/− newborns. (A,B) Diaphragmatic herniation in IIA^+/− IIB^−/− newborn. The stomach and spleen are located in the abdominal cavity, below the diaphragm in wild-type or IIB^−/− mice (A). In some IIA^+/− IIB^−/− mice, the stomach and hypoplastic spleen were mislocated above the diaphragm (B). (Inset) The stomach and spleen located behind the lung. D, diaphragm; L, lung; Lv, liver; St, stomach. (C,D) Body wall herniation in E18.5 IIA^+/− IIB^−/− fetus (D). Arrows indicate region of the umbilical ring. (E–H) Lateral view of a wild-type (E) and three IIA^+/− IIB^−/− (F–H) newborn pups, displaying variable defects in anterior head, cyclopia, and tail formation. (F,G insets) The frontal view of the corresponding mutants. (I–L) Cleft palate (J) and bilateral kidney agenesis (L) in IIA^+/− IIB^−/− mutant mice. The arrows in J indicate cleft palate. A, adrenal gland; B, bladder; K, kidney; O, ovary; Ut, uterine; U, urethra.

Figure 3

Gdf11 binds to IIA and IIB and phosphorylates Smad2. (A) Gdf11 can induce Smad2 phosphorylation. Activated Smad2 and suppressed Smad1 phosphorylation were detected by anti-phospho-Smad1 (α-PSmad1) or anti-phospho-Smad2 antibodies (α-PSmad2) in stage 10 ectodermal explants of Xenopus embryos. Cytoskeletal actin was used as a loading control (α-Actin). (B) Both Gdf11 and Flag–Gdf11, but neither Gdf10 nor Flag–Gdf10, induced Smad2 phosphorylation, indicating that Flag–Gdf11 is functionally active. (C) Coimmunoprecipitation analyses showing that Gdf11, but not Gdf10, interacts with the activin receptor complexes (top panel). Note that IIA coprecipitated with ALK4 effectively only in the presence of Gdf11, whereas IIB did so regardless of the ligand (second panel). Comparable levels of protein expression in total extracts are shown. (D) Coimmunoprecipitation analyses showing that Gdf11 binds to IIA and IIB to different degrees. Both IIA and IIB were coprecipitated with Gdf11 in the absence or presence of ALK4 (top panel), whereas ALK4 was coprecipitated with Gdf11 only in the presence of type II receptors (fourth panel). Note that the amount of Gdf11 coprecipitated with IIA was smaller than that with IIB (top panel). For all experiments, 2 ng of IIA(KR)–Myc or IIB(KR)–Myc mRNA was injected, except for the last lane in which 1 ng each of IIA and IIB mRNA was injected instead.