Volume 11, Issue 3, Pages 313-323 (September 2006) The Nodal Precursor Acting via Activin Receptors Induces Mesoderm by Maintaining a Source of Its Convertases and BMP4  Nadav Ben-Haim, Cindy Lu, Marcela Guzman-Ayala, Luca Pescatore, Daniel Mesnard, Mirko Bischofberger, Felix Naef, Elizabeth J. Robertson, Daniel B. Constam  Developmental Cell  Volume 11, Issue 3, Pages 313-323 (September 2006) DOI: 10.1016/j.devcel.2006.07.005 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 The Nodal Precursor Induces Epithelial-Mesenchymal Transitions Marked by Downregulation of E-Cadherin (A–C) Sagittal sections of NodalNr/+ heterozygotes (A), NodalNr/Nr homozygotes (B), and Nodal null mutants (C) at E7.5. Whereas Nodal null mutants fail to gastrulate (C), Nr/Nr homozygotes convert the entire epiblast into mesenchymal tissue (B). (D–F) In normal embryos, expression of Snail1 marks ectoplacental cone (epc) and cells in the primitive streak (ps) undergoing an epithelial-mesenchymal transition (D). In contrast, NodalNr/Nr homozygotes ectopically express Snail1 in the rudimentary chorion (asterisk) and throughout the embryonic region (E). In Nodal null mutants, lack of Snail1 mRNA confirms the absence of a primitive streak (F). (G) Expression of E-cadherin in the extraembryonic ectoderm and epiblast. (G′) Boxed area of (G) at higher magnification shows that apical expression of E-cadherin (filled triangles) is reduced in the primitive streak region (open triangles). (H and H′) Inhibition of Nodal processing in NodalNr/Nr mutants blocks expression of E-cadherin in the epiblast. (I and I′) The epiblast in Nodal null mutants retains significant E-cadherin expression. al, allantois; am, amnion; ch, chorion; epi, epiblast; m, mesoderm. Size bars: 50 μm. Developmental Cell 2006 11, 313-323DOI: (10.1016/j.devcel.2006.07.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Disruption of the SPC Recognition Motif in the Nodal Precursor Blocks the Specification of AVE and Mesendoderm while Stimulating Ectopic Mesoderm Formation (A) Wild-type Nodal mRNA is expressed at E5.5 in the epiblast and embryonic visceral endoderm; by E6.5, it localizes to the nascent primitive streak in the posterior epiblast. Nodal signaling specifies anterior visceral endoderm (AVE) marked by the expression of Hesx-1, Cer1, Lhx1, and Gsc. At the opposite pole, Lhx1 and Gsc transcripts also mark mesendoderm progenitor cells in the epiblast (top panels). In NodalNr/Nr homozygotes, Nr mRNA was undetectable at E5.5. Even though transient expression of Nr was detected at E6.5 in the proximal and medial epiblast, markers of AVE and mesendoderm fail to be induced (bottom panels). (B) Nr homozygotes also fail to express the Nodal coreceptor Cripto, whereas posterior mesoderm markers including Wnt3, Brachyury, and Fgf8 mRNAs are present. However, Brachyury mRNA expression was never detected in the ExE (above stippled line) between E5.5 to E6.5 and by E7.5, is prematurely lost in the epiblast. Likewise Lefty-2, a direct target gene of Nodal in the primitive streak fails to be induced. Size bars: 50 μm. Developmental Cell 2006 11, 313-323DOI: (10.1016/j.devcel.2006.07.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 SPC-Resistant Nodal Stimulates Mesoderm Formation by Maintaining the Extraembryonic Source of Bmp4, which in Turn Induces Wnt3 (A) Expression of Wnt3 and Brachyury mRNAs is lost in epiblast explants cultured without visceral endoderm (green) and ExE (gray), irrespective of the presence or absence of processed Nodal or cleavage-resistant precursor (Nr). In contrast, treatment with recombinant BMP4 restores expression of Wnt3 and Brachyury. Brachyury expression is also rescued by the Wnt3 ortholog Wnt3A. (B) Whereas Nodal null mutants (lacZ/lacZ) downregulate Bmp4 expression in the ExE (Beck et al., 2002; Brennan et al., 2001; Guzman et al., 2004) irrespective of the genetic background, it is maintained in NodalNr/Nr homozygotes. Localized expression of the Bmp4 target gene Fragilis/Ifitm3 in the allantoic bud and proximal epiblast confirms the presence of active Bmp4 in wild-type and NodalNr/Nr embryos. By contrast, Fragilis is diffusely expressed in Nodal null mutants. Size bars: 50 μm. Developmental Cell 2006 11, 313-323DOI: (10.1016/j.devcel.2006.07.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Wnt3 Signaling Maintains Expression of Nodal through the Proximal Epiblast-Enhancer (A–P) Wild-type (A, B, E, F, I, J, M, and N) and Wnt3−/− embryos (C, D, G, H, K, L, O, and P) induce the NodallacZ reporter allele (A–D), the ASE-lacZ transgene (E–H), and a mutant derivative of the NodallacZ reporter devoid of the Nodal-responsive ASE enhancer (I–L). However, expression of these reporter genes does not reach normal levels at E6.0 and fails to be maintained at E6.5 in Wnt3 mutants. Activity of the proximal epiblast enhancer (PEE) of Nodal monitored by a PEE-lacZ reporter transgene (M and N) is undetectable in Wnt3−/− embryos (O and P). (Q) Conservation of two Tcf consensus motifs (CCTTTGA) in the mouse PEE element 12.1 kb upstream of the start codon. In rat and armadillo, the precise distances are unknown due to gaps in the available genome databases. (R) Isolated epiblast explants fail to maintain expression of Nodal. (S) Nodal expression is rescued in epiblast explants incubated with Wnt3A. Size bars: 50 μm. Developmental Cell 2006 11, 313-323DOI: (10.1016/j.devcel.2006.07.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 The Nodal Precursor Amplifies the Expression of Its Proprotein Convertases Furin and PACE4 in the ExE (A) PACE4 (left column) and Furin (right column) are expressed in the ExE of control and NodalNr/Nr embryos but downregulated in Nodal null mutants (lacZ/lacZ). (B) PACE4 and Furin expression in ExE explants cultured in the presence or absence of recombinant Nodal (Ndl) or Nr. Size bars: 50 μm. Developmental Cell 2006 11, 313-323DOI: (10.1016/j.devcel.2006.07.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Activin Receptors Mediate SPC-Independent Nodal Signaling in the ExE and Bind Purified Nodal Precursor Protein (A) Ten micromolar SB431542, a pharmacological inhibitor of type I activin receptors, blocks induction of Bmp4 and PACE4 in ExE explants treated with purified recombinant Nr. Size bar: 50 μm. (B) The extracellular domain of activin receptor IIB specifically coimmunoprecipitates with anti-Flag beads coated with purified Nr but not with empty beads. (C) After chemical crosslinking of cell surface proteins, HA-tagged ALK4 (arrow) specifically coprecipitates with cotransfected Nr and to a lesser extent with constitutively active, mature Nodal expressed without a pro domain (Nmat). Traces of a 33 kDa form of Nmat presumably correspond to incompletely reduced dimer. The arrowhead denotes a nonspecific IgG band. Kinase-inactive mutants (KR) of ALK4 and ActRIIB were used to attenuate endocytosis. (D) Nr binds to and does not inhibit the assembly of an ALK4/ActRII complex at the surface of transfected 293T cells. Developmental Cell 2006 11, 313-323DOI: (10.1016/j.devcel.2006.07.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 The Nodal Precursor Activates Two Interlinked Feedback Loops, which Regulate Mesoderm and Mesendoderm Formation (A) SPC-independent Nodal signaling from the epiblast (blue) to the ExE (gray) is sufficient to sustain expression of Bmp4, which specifies posterior mesoderm at E6.5 through induction of Wnt3. In addition, Wnt3 mediates positive feedback by stimulating the proximal epiblast enhancer (PEE) of Nodal. In ExE cells (gray), the Nodal precursor also maintains the expression of its convertases Furin and PACE4, which stimulate Nodal autoinduction via the asymmetric enhancer (ASE) to generate the Cripto-dependent high threshold Nodal signal required for mesendoderm formation. Since Furin can also cleave proBmp4 (stippled arrow), this explains why Furin−/−;PACE4−/− double mutants apparently form less ectopic mesoderm than NodalNr/Nr embryos. Negative feedback loops limiting the range of Nodal signaling to prevent excess endoderm and mesoderm formation are not depicted. For the in silico analysis, the integral I of Nodal precursor N (proN[t]) over time, weighted by the population size P(t) secreting Nodal, is used as a measure of Nodal signaling strength. (B) Only a relatively slow Wnt3-mediated feedback loop significantly boosts the Nodal-signaling strength I by maintaining Nodal activity. A two loop model (Brandman et al., 2005) is used to compute for varying Wnt3 loop velocities τWnt3 how the Nodal integral I develops in time. The matN loop mediated by mature Nodal is kept constant, with τWntN = 720E−1. (C) A Nodal-signaling network with combined fast and slow loops can account for the cell fates observed in wild-type, Nr mutants, and Wnt3 mutants. The velocity of the slow loop in the wild-type was chosen such that the Nodal signal decays no later than around E7.5, as observed experimentally (red curve in [B]). The integrated Nodal signal is shown in function of time for three genotypes: wild-type (the two loop model), Wnt3−/− (a single fast loop), and NodalNr/Nr (a single slow loop). Parameters are identical for all genotypes (Table S2). Developmental Cell 2006 11, 313-323DOI: (10.1016/j.devcel.2006.07.005) Copyright © 2006 Elsevier Inc. Terms and Conditions