Genikhovich G , Fried P , Prünster MM , Schinko JB , Gilles AF , Fredman D , Meier K , Iber D , Technau U .

P 22717 Austrian Science Fund FWF, P 26962 Austrian Science Fund FWF

	Figure 1. BMP Signaling Is Strongest on GDF5-like-Expressing Side of Nematostella Embryo(A) Positions of chordin (blue) expression, bmp4 (green) expression, and BMP signaling domain (red circles) in different animal models.(B and C) Schematic representation of NvDpp, NvBMP5-8, NvGDF5-like, NvChd, NvGrm, NvGbx, and Hox expression domains in planula viewed laterally and orally. Red lines, cutting planes; black double-headed arrows, directive axis; asterisks, blastopore.(D–K) The αpSMAD1/5 and αNvHoxE antibody staining in control and morphant early planulae, n > 50 for each sample; (D–I) lateral views; (J and K) oral views; asterisks, blastopore. (D) αpSMAD1/5-positive nuclei are located on NvHoxE-expressing side. αpSMAD1/5 and αNvHoxE stainings partially overlap. (E–K) αpSMAD1/5 and αNvHoxE in StdMO, ChdMO, GrmMO, GDF5lMO, BMP5-8MO, and DppMO embryos. Staining is absent in DppMO, BMP5-8MO, and ChdMO and suppressed (white arrow) in GDF5lMO (E–H); the domain showing strong staining (white double-headed arrows and white demarcating lines) is narrower in the StdMO than in the GrmMO (J and K).See also Figure S1.
	Figure 2. Effect of MO Knockdown on αpSMAD1/5 Staining Gradient and Morphology of Late Planulae(A–D) Nuclear αpSMAD1/5 staining intensity as a function of relative position of each endodermal nucleus along 180° arc from 0 to π starting at the middle of pSMAD1/5-positive domain (see the Supplemental Experimental Procedures and Figure S2A for details on measuring), described by measurements from multiple embryos (colored points) for Control (n = 12), GrmMO (n = 7), GDF5lMO (n = 10), and DppMO (n = 8). LOESS smoothed curves (black lines) with 99% confidence interval for the mean (shade), and linear model fit of the logarithm of relative position to pSMAD1/5 staining intensity (R2 = 0.73, dashed red line) for Control embryos and linear model fit of relative position to pSMAD1/5 staining intensity (R2 = 0.55, dashed red line) for GrmMO embryos. Oral views of corresponding StdMO, DppMO, GDF5lMO, and GrmMO late planulae stained with fluorescent phalloidin are shown next to the graphs (n > 55 for each sample). While StdMO planulae with normal nuclear pSMAD1/5 gradient develop eight mesenteries (red arrows in A), DppMO-injected planulae lacking the pSMAD1/5 gradient do not develop mesenteries at all (B). αpSMAD1/5 staining intensity is suppressed but not absent in the GDF5lMO planulae correlating with the formation of four mesenteries instead of eight (red arrows in C). In GrmMO-treated embryos (D), the average pSMAD1/5 staining intensity is elevated in mid-range positions (see also Figure S2C) and more variable than in Control embryos across the whole range (see also Figure S2D). The endoderm in GrmMO appears compartmentalized but its development is abnormal with extremely thickened mesogloea (green arrow). Scale bars represent 50 μm.See also Figure S2.
	Figure 3. Staggered Endodermal Expression of Hox Genes and NvGbx Is Regulated by BMP Signaling(A–DD) Expression of NvHoxB (A–F), NvHoxD (G–L), NvHoxE (M–R), NvHoxF (S–X), and NvGrm (Y–DD) in StdMO-, BMP5-8MO-, DppMO-, GDF5lMO-, ChdMO-, and GrmMO-injected embryos at early planula stage. Lateral views; oral end to the left. Embryos on (Y) and (DD) are rotated 90° compared to other stained embryos to make bilateral expression of NvGbx visible.(EE–HH) Ectopic expression of BMPRIQ209 > D-EGFP in patches of ectodermal cells in 2-day planula results in ectopic activation of NvHoxE protein. (EE) Ectopic pSMAD1/5-positive nuclei in the ectoderm of early planula in addition to endogenous staining in the endoderm (blue dots). (FF) In addition to endogenous αNvHoxE staining (blue dots), ectopic expression is observed in pSMAD1/5-positive ectodermal nuclei. Apart from specific nuclear NvHoxE signal, the secondary anti-rat antibody cross-reacts with mouse anti-GFP antibody staining the BMPRIQ209 > D-EGFP fusion protein in the cell membranes. (GG) Anti-GFP antibody detects BMPRIQ209 > D-EGFP in cell membranes. Anti-mouse antibody does not cross-react with the rat αNvHoxE, thus no nuclear staining is observed, showing that nuclear staining on (FF) is specific for NvHoxE. (HH) Merged image of (EE–GG). White dots indicate the border between ectoderm and endoderm. White arrows point at two strongly pSMAD1/5-positive ectodermal nuclei in transgenic cells, which also ectopically express NvHoxE. Yellow arrows point at a weakly pSMAD1/5-positive ectodermal nucleus in a transgenic cell, which does not express detectable amounts of NvHoxE. Scale bars represent 50 μm.See also Figure S3.
	Figure 4. Maintenance of the Directive Axis in Nematostella(A) Expression analysis of network components in morphant early planulae; lateral views, oral end is to the left. Scale bars represent 50 μm.(B) Mathematical model correctly predicts expression patterns of all genes shown in (A) (color code as in ISH), as well as pSMAD1/5 localization (shown as heat maps) in the knockdowns. All expression patterns and pSMAD1/5 staining patterns are plotted onto mesogloea and shown as 2D projections of a 3D structure. Expression domains of NvDpp/NvBMP5-8/NvChd, although not identical, strongly overlap when plotted onto mesogloea. In the model they are controlled by the same mathematical expression and thus plotted together.See also Figures S3 and S4.
	Figure 5. Strongly Constrained Core Interactions and Weakly Constrained Modulators of the BMP Signaling System(A and B) Nematostella directive axis network (A) compared to the DV network in Xenopus (B). Blue lines represent transcriptional regulation downstream of BMP signaling and black lines represent protein-protein interactions (putative in case of Nematostella). Xenopus network is modified after (De Robertis and Colozza, 2013; Paulsen et al., 2011). Overlaid blue and black lines represent shuttle function of Chordin.(C–E) Restricted chordin expression domain (red curves on the left plots) results in BMP signaling on the opposite side of the embryo (relative concentration of BMP ligand-receptor complexes shown as black curves on Cʹ–Eʹ) independent of bmp4 expression localization (blue curve). bmp4 can be expressed uniformly (C), on the chordin side (D), or opposite to it (E). The x axis is normalized with respect to maximal domain length (shown as 2π since the embryo is spherical), and the y axis shows the ratio of ligand-bound receptors (LR) with respect to total receptor concentration (RT).(F and G) Constrained core regulators versus weakly constrained modulators of BMP signaling in Nematostella (F) and Xenopus (G).(F) Many parameters describing production, function, and turnover of NvDpp/NvBMP5-8 and NvChd (blue circles) are strongly constrained, while most of the parameters describing production, function, and turnover of NvGDF5-like and NvGrm (orange circles) are weakly constrained. Large orange circle with a 9 corresponds to nine unconstrained parameters related to NvGDF5-like and NvGrm.(G) Many parameters describing production, function, and turnover of frog BMP4/BMP7, ADMP/BMP2, Chordin, and Tolloid (teal circles) are strongly constrained, while most of the parameters describing production, function, and turnover of BAMBI and SMAD6/7 (pink circles) are weakly constrained. Large pink circle with an 8 corresponds to eight unconstrained parameters related to BAMBI and SMAD6/7. Fold of possible increase of parameter values (log scale) is plotted on the y axis, and fold of possible decrease of parameter values (log scale) is plotted on the x axis. (For details see the Supplemental Experimental Procedures and Figure S5.)See also Figure S5.

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