Reissmann E , Jörnvall H , Blokzijl A , Andersson O , Chang C , Minchiotti G , Persico MG , Ibáñez CF , Brivanlou AH .

EY12370 NEI NIH HHS , HD32105 NICHD NIH HHS , R01 HD032105 NICHD NIH HHS

	Figure 1. Functional characterization of ALK7 by gain-of-function experiments in Xenopus laevis. (A) Morphology of animal caps expressing the constitutively active mutant form of rat ALK7 (CA-ALK7). Embryos were injected in the animal pole at the two-cell stage, animal cap explants were dissected at the blastula stage and allowed to develop until sibling control embryos reached tailbud stage 25. (B) Dose-response induction of mesendodermal marker genes in animal caps injected with increasing concentrations of CA-ALK7. Embryos were injected at the two-cell stage, animal cap explants were dissected at the blastula stage, and harvested at stage 25. EF1α is shown as loading control. (−RT) No reverse transcriptase; (E) total embryo; (un) uninjected control explants. Data are representative of four independent experiments. (C) CA-ALK7 and CA-ALK4 induce early mesendodermal marker genes in a dose-responsive manner. RT–PCR analyses took place at the gastrula stage. (D) CA-ALK7 and CA-ALK4 both elicit late mesodermal marker genes, but show differences in their ability to maintain expression of Xnr genes in ectodermal explants. RT–PCR analyses were performed at the tailbud stage (stage 20).
	Figure 2. Role of ALK7 in cell fate and cell localization. (A–C) Control embryos injected with LacZ RNA. The β-gal staining at stage 45 is restricted to skin. (A) Dorsal view. (B) Ventral view. (C) Higher magnification of ventral view with focus on anterior embryonic regions. (D–G) Embryos were co-injected with CA-ALK7 mRNA and LacZ mRNA. The β-gal staining detects descendants of the injected ectodermal cells specifically localized to the gut, indicating an ectoderm-to-endoderm cell fate transformation and translocation. (D) High magnification with focus on the gut region. (E) Dorsal view. (F) Lateral view. (G) Ventral view.
	Figure 3. Dominant-negative ALK7 (DN-ALK7) produces anterior defects and cyclopia, and inhibits Xnr1 activity in ectodermal explants and in whole embryos. (A) Embryonic phenotype of DN-ALK7 overexpression in vivo. Injection of 2 ng DN-ALK7 into all four marginal cells at the eight-cell stage resulted in anterior head defects, including cyclopia, and axial truncations (19% and 87% respectively, n = 46). Control sibling at late tadpole stage. Anterior to the left. (B) DN-ALK7 inhibits the formation of a partial secondary axis by Xnr1. Injection of 1 ng Xnr1 or Xnr2 RNA into one ventral-vegetal cell at the eight-cell stage resulted in a partial secondary axis (62%, total n = 32; 54%, total n = 22, respectively). Co-injection of 4 ng DN-ALK7 RNA inhibited the Xnr1-mediated phenotype (92%, totaln = 28), whereas Xnr2 activity was unaffected. A sibling control embryo (stage 28) is shown in the bottom panels.
	Figure 4. DN-ALK7 and DN-ALK4 inhibit mesoderm formation induced by Xnr1 and Nodal, whereas the activities of Xnr2, Xnr4, and Activin are only efficiently inhibited by DN-ALK4. (A) DN-ALK7 inhibits late and early mesodermal marker gene expression induced by Xnr1. DN-ALK7 alone did not induce any marker genes (lane 9). (un) Uninjected explants; (E) whole embryo control; (−RT) no reverse transcriptase. Stage 25 (late) and stage 11 (early) were analyzed. (B) Dose-response analysis of mesoderm-inducing ligand activity in the absence or presence of DN-ALK7 and DN-ALK4. (Lanes1–4) Ligand (RNA concentrations for Xnr1, Xnr2, Xnr4, and Nodal were 1 ng, 0.5 ng, 0.1 ng, and 0.05 ng, respectively; RNA concentrations for Activin were 10 pg, 5 pg, 1 pg, and 0.5 pg, respectively); (lanes 5–8) ligands in the presence of 2 ng DN-ALK7; (lanes 9–12) ligands in the presence of 2 ng DN-ALK4; (lanes 13,14) DN-receptors alone.
	Figure 5. Reconstitution of functional receptors for Xnr1 and Nodal in heterologous cells. (A) Transactivation of ALK7 by overexpression of ActRIIB, but not ActRIIA or BMPRII, in HepG2 cells. The dose of CA− or wild-type (wt) ALK7 plasmid DNA was 1 ng per three wells. Type II receptors were transfected at 0.1, 0.3, 1, and 3 ng per three wells. (B) ALK7 and ActRIIB confer responsiveness to Xnr1 (left) and mouse Nodal (right) in heterologous cells. Ligand DNAs were transfected at the indicated amounts (per three wells). Results are expressed as mean ± SD, and are representative of three independent experiments. (C) ALK4 and ActRIIB do not suffice to confer responsiveness to Xnr1 and Nodal. HepG2 cells were transfected with ALK4, ActRIIB, and the indicated ligands. Results are expressed as mean ± SD, and are representative of three independent experiments. (D) Cripto enhances the response of ALK7 to Nodal proteins and allows Nodal and Xnr1 signaling via ALK4. HepG2 cells were transfected with receptors and ligands as indicated. In the absence of transfected ALK4 or ALK7 receptors, Cripto produced a much smaller response to Xnr1 and Nodal (Cripto alone), in agreement with the presence of low levels of endogenous Activin receptors in HepG2 cells. Results are expressed as mean ± SD, and are representative of three independent experiments.
	Figure 6. ALK7, ActRIIB, and Cripto can bind Xnr1 directly, whereas ALK4 and ALK7 can associate with Cripto. (A) Binding of Xnr1 to soluble ALK7-Fc. Residual Activin signal in the ALK7-Fc lane is comparable with control, indicating background binding. (B) Binding of Xnr1 to soluble ActRIIB-Fc. As control, a purified GFRα1-Fc fusion was used (control Fc). (GFRα1 stands for glial cell line-derived neurotrophic factor (GDNF) family receptor α-1). (C) Cripto associates with ALK4 in the absence of ligand. Following precipitation with Protein-G beads, Cripto was detected by immunoblotting with anti-Histidine antibodies. Comparable amounts of Fc fusion proteins were used as determined by re-probing with anti-human IgG antibodies. (D) Reverse pull-down of ALK4-Fc and Cripto. Following precipitation with metal-affinity beads, ALK4-Fc was detected by immunoblotting with anti Fc antibodies. (E) Cripto associates with ALK7. Co-precipitation assay showing binding of purified ALK7-Fc to purified His-tagged Cripto. Following precipitation with metal-affinity beads, ALK7-Fc was detected by immunoblotting with anti-Fc antibodies. (F) Cripto binds Xnr1 directly. Co-precipitation assay showing binding of HA-tagged Xnr1 produced in COS cells to purified His-tagged Cripto. Following precipitation with metal-affinity beads, Xnr1 was detected by immunoblotting with anti-HA antibodies.
	Figure 7. ALK7 mRNA is localized in the ectodermal and organizer regions of Xenopus gastrula embryos and is expressed during early stages of mouse embryonic development. (A) Amino acid sequence alignment of Xenopus and rat ALK7 fragments. The Xenopus ALK7 fragment obtained overlaps with the rat sequence from amino acid residues 259 to 312 located to the kinase domain. (+) Conservative changes; (−) non-conservative changes. (B) Phylogenetic analysis of homologous fragments from Xenopus and rat ALK7, and the related receptors ALK4 and ALK5. The tree was drawn using NJPLOT based on an alignment made in CLUSTALX. (C) Xenopus ALK7 is expressed maternally and persists throughout development as analyzed by RT–PCR on oocytes and embryos. (D) Expression of xALK7 is localized to dorsal-animal regions in early gastrula stage embryos. As control for correct embryo dissection the following marker genes were used: (Gsc) goosecoid (dorsal expression); (Xbra) Brachyury (dorsal–ventral or marginal); (msx) muscle-segment-homeobox-related gene (ventral); (EK) epidermal keratin (animal-ventral). Histone was used as loading control. (E) In situ hybridization analysis reveals expression of ALK7 in the organizer. Cross sections of those embryos show ALK7 expression in dorsal (d) and animal pole region (an). The blastopore lip (bp) is indicated. (v) Ventral; (veg) vegetal. (F) Expression of ALK7 mRNA in the early mouse embryo. RNA from the indicated days of mouse embryonic development (lanes1–4), as well as adult brain mRNA (lane 5) and negative control (yeast tRNA, lane 6) were analyzed by RNase protection assay (RPA) for expression of ALK7 mRNA using a specific mouse ALK7 riboprobe.
	acvr1c (activin A receptor, type IC) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, sagittal section, animal up, dorsal left.

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