Reversade B , De Robertis EM .

R37 HD021502-19 NICHD NIH HHS , R37 HD21502-19 NICHD NIH HHS , R37 HD021502 NICHD NIH HHS

	Figure 1. An Assay for Self-Regulative Development: DV Patterning of Dorsal Half-Embryos Is Independent of BMP4/7 Signals (A) Embryos were injected with a mixture of Bmp4/7 MOs (12 ng each) and bisected at blastula stage (n = 15 or more per experimental set). (B) Uninjected control sibling. (C) Dorsal halves self-regulate. (D) Ventral halves develop into a ventralized belly-piece lacking all CNS. (E) Dorsal half injected with Bmp4/7 MOs self-regulates pattern. (F) Ventral halves lacking BMP4/7 activity elongate and differentiate Sox2-positive CNS. (G and H) Multiple ventral half-embryos illustrate reproducibility of the assay.
	Figure 2. Admp Expression Is Triggered by Low BMP Signaling, and Admp MO Dorsalizes the Embryo. (A) The Xenopus ventral and dorsal centers. (B) Admp is expressed dorsally (stage 12). (C) Chordin protein injection in the blastula cavity (5 ng) leads to upregulation of Admp expression. (D) Admp MO targeting both Xenopus laevis Admp pseudoalleles. (E) Xenopus Admp translation is specifically inhibited by Admp MO but not by control MO of random sequence. (F) ADMP causes phosphorylation of endogenous Smad1 in Xenopus embryos, which is blocked by Chordin protein injection.(G and H) Admp MO-injected embryos (12 ng total) are dorsalized.(I) zAdmp mRNA (75 pg total) ventralizes the Xenopus embryo.(J) Admp MO rescued by zAdmp mRNA.
	Figure 3. Self-Regulative DV Patterning in Dorsal Half-Embryos Requires ADMP and Has the Opposite Transcriptional Regulation of BMP4 and Ventral BMP Antagonists. (A) Experimental design (n = 28 or more per experimental set). (B) Sox2 expression in uninjected siblings. (C) Control blastula dorsal halves self-regulate. (D) The ventral half forms belly-pieces lacking CNS. (E) In dorsal halves injected with Admp MO, much of the ectoderm becomes CNS. (F) Ventral half-embryos are unaffected by Admp MO. (G) RT-PCR analysis of whole embryos at stage 11 after injection of Chordin (1 and 5 ng) or BMP4 (0.5 and 2.5 ng) protein (in a volume of 40 nl) into the blastocoele cavity at stage 8. (H) Sox2 expression in control embryos at stage 20. Inset illustrates the dorsal and ventral centers marked by Chordin and Sizzled at stage 12. (I) Coinjection of Bambi and Sizzled MOs ventralizes the embryo. (J) A single dorsal injection of xAdmp mRNA (50 pg) into wild-type embryo decreases anterior CNS differentiation and increases Sizzled expression (inset). (K) Depletion of Bambi and Sizzled greatly sensitizes the embryo to the effects of Admp mRNA. (L) Model of regulatory interactions in the Xenopus embryonic field. Green lines indicate pro-BMP effects; red lines indicate anti-BMP effects
	Figure 5. ADMP and Chordin Interact In Vivo (A) Epistasis experiment in embryos injected with Admp MO, Chordin MO, or a mixture of both (n = 23 or more per experimental set). (E) Injection of xAdmp mRNA (25 pg in each blastomere at the four-cell stage) abolishes CNS formation (absence of Sox2 expression). (F) A single dorsal injection of Chordin mRNA (50 pg) rescues CNS formation in embryos ventralized by Admp mRNA injection. (G) Secondary axes (marked by Sox2, n = 37) induced by a single ventral injection of Chordin mRNA (50 pg) lack eye and forebrain tissues (marked by Six3, inset). (H) In ADMP-depleted embryos, Chordin mRNA induces complete secondary axes including eye and forebrain tissues (n = 21). (I) Spemann organizer grafts at midgastrula (stage 103⁄4 ) induce trunks with no eyes (n = 9) or cyclopic eyes (n = 4). (J) ADMP-depleted Spemann organizer grafts transplanted at midgastrula (stage 103⁄4 ) induce complete head structures with two eyes (in 7 of 13 grafts).
	Figure 6. Quadruple Knockdown of BMP2/4/7 and ADMP Results in Ubiquitous CNS Formation In Vivo (A) CNS marked by Sox2 in control embryos; inset shows frontal view (DAI = 5.2). (B) Knockdown of ADMP moderately increases Sox2 expression in anterior CNS (inset) (DAI = 6.2). (C) Triple knockdown of BMP2/4/7 results in embryos with a neural plate approximately 5 times the width of that of control siblings (transversal section shown in inset). (D) Quadruple inactivation of BMP2/4/7 and ADMP (9 ng total each) results in radially dorsalized embryos and ubiquitous neural differentiation in the ectoderm (DAI = 9.7; n = 59). (E) Cytokeratin expression marks epidermal cells on the surface of control embryos. (F) Expression of epidermal Cytokeratin is abolished in Bmp2/4/7/Admp morphants at stage 16. (G and H) BMP2/4/7/ADMP-deficient embryos express radial Otx2, Rx2a, and Krox20. (I) Quadruple Chordin/Noggin/Follistatin/Cerberus MO-injected embryos are strongly ventralized (DAI = 2.3) but retain posterior CNS (compare to [K]). (J) ADMP is held inactive by forming a stable inhibitory ternary complex with Chordin and Tsg. DN-Xlr inhibits Xolloid-related (Xlr), preventing the proteolytic cleavage of Chordin at two specific sites. (K) Sox2 in wild-type embryos. (L) Injection of DN-Xlr mRNA (400 pg) broadens Sox2 expression. (M) Simultaneous knockdown of BMP2/4/7/ADMP and of Chordin/Noggin/Follistatin/Cerberus display ubiquitous neural induction, indicating that BMP ligands are epistatic to their secreted antagonists. (N) Blastocoele injection (40 nl) of Noggin-Fc (0.5 mM) or Chordin (1 mM) protein causes complete neuralization of ectoderm. (O) BMP2/4/7-depleted embryos retain nonneural ectoderm. (P) Ubiquitous neural differentiation in BMP2/4/7-depleted embryos injected with DN-Xlr mRNA; this experiment suggests that Chordin cleavage by Xlr is required for ADMP-mediated epidermal differentiation.
	Figure 7. Rescue of BMP2/4/7/ADMP-Depleted Embryos by BMP4 Protein and Demonstration that Ventral and Dorsal Centers Serve as BMP Sources In Vivo. (A) Bmp2/4/7/Admp MOs neuralize the entire ectoderm (n = 25 or more per experimental set). (B) Epidermal fate and DV pattern are rescued in BMP2/4/7/ADMP-depleted embryos by injection of recombinant BMP4 protein (1.5 ng in 40 nl). (C) Experimental design of blastomere lineage-tracing experiments. (D) B4 blastomere descendants are distributed along the AP axis of BMP2/4/7/ADMP-depleted embryos (n = 17). (E) DV pattern in BMP2/4/7/ADMP-depleted embryos is rescued by BMP4 protein injection (n = 56) according to the initial DV polarity of the fertilized egg, as demonstrated by nuclear LacZ staining in the B4 lineage. (F) Experimental procedure in which wild-type grafts (marked by nuclear lacZ) were transplanted into ADMP/BMP2/4/7-depleted hosts at early gastrula. (G) Ventral-center grafts (inset) rescue neural plate and epidermal differentiation over many cell diameters (n = 19; for control nongrafted hosts, see [A] and Figure 6F). This experiment demonstrates that the ventral center produces organizing signals. (J) Dorsal-center grafts elongate and rescue neural and epidermal patterning over the entire embryo (n = 21). The dorsal graft is the sole source of BMP in these quadruple BMP2/4/7/ADMP-depleted hosts. Note that epidermis is induced only at a distance.
	Figure S1. Comparative Expression of Admp and Chordin during Early Xenopus Development. (A and A' Chordin expression is first detected in superficial cells at the blastula stage in a dorso-animal domain (which spans animal cap and marginal zone cells) corresponding to the BCNE (Blastula Chordin and Noggin Expressing) Center (Kuroda et al., 2004). The BCNE center gives rise to brain, floor plate and notochord at later stages of development. The dotted line indicates the direction of the section shown in the right panel. (B and B Admp expression is detected at stage 9 in a similar dorso-animal domain, but in cells located deeper than the ones expressing Chordin. (C and C At stage 11, expression of Chordin expands and is seen in involuted cells of the future prechordal plate (pp) and midline. (D and D At a similar stage, Admp is also expressed locally in involuting cells of the prospective midline, but in a narrower and more posterior domain than that of Chordin. (E and E At stage 13, Chordin is expressed in a broad domain within the prechordal plate (pp) and in notochord (no). (F and F Admp expression at stage 13 is localized to the floor plate (fp), a superficial layer of midline cells overlying the notochord. (G and G Expression of Chordin at early neurula stage persists in the notochord (no) and is turned off in the prechordal plate. (H and H At early neurula stage, Admp expression fades and is seen only in the posterior-most region of the floor plate. (I and J) At tadpole stage, Chordin expression is restricted to the posterior notochord, while expression of Admp is no longer detectable.
	Figure S2. Xenopus Admp Overexpression Activates Known BMP Target Genes (A) Vent-1, a direct target of BMP signaling, is expressed in the ventral part of the closing blastopore at stage 12. (B) Xenopus Admp mRNA injection (25 pg in each blastomere at the 4-cell stage) up-regulates Vent-1 expression over the entire surface of the embryo. (C) Bmp4 expression is localized at stage 12 in the ventral part of the closing blastopore (or ventral center). (D) Admp mRNA overexpression turns on Bmp4 transcription uniformly indicating that Bmp4 is under positive feedback regulation by BMP-like signals. (E) MyoD, which marks future somites, is expressed at stage 12 as a mesodermal ring around the closing blastopore with exception of the future notochord. (F). Admp mRNA overexpression eliminates notochord differentiation leaving a radial ring of MyoD expression around the closing blastopore. These experiments indicate that ADMP has similar phenotypic effects to those of other BMP signals.
	Figure S3. Chordin/Admp and Sizzled/Bambi mark the dorsal and ventral center and are inversely regulated by BMP levels. (A) The dorsal center or Spemann organizer is marked by Admp expression while the ventral center is marked by Bambi expression. (B) Low BMP levels (achieved by injecting recombinant Chordin protein in the blastocoele cavity) expand Admp expression in the Spemann organizer at the expense of Bambi expression in the ventral center. Admp and Bambi are under opposite transcriptional regulation by the BMP pathway. (C) High BMP levels (achieved by injecting recombinant BMP4 protein in the blastocoele cavity) reduce Admp expression in the Spemann organizer while augmenting Bambi expression in the ventral center. When Admp expression goes down, Bambi expression goes up and vice versa. (D) The dorsal center or Spemann organizer is marked by Chordin expression while the ventral center is marked by Sizzled expression. (E) Low BMP levels expand Chordin expression in the Spemann organizer at the expense of Sizzled expression in the ventral center. Chordin expression is activated by low BMP levels while Sizzled expression is repressed. (F). High BMP levels reduce Chordin expression in the Spemann organizer, while increasing Sizzled expression in the ventral center. When Chordin expression decreases, Sizzled expression increases.
	Figure S6. Loss of ADMP Renders the Embryo Hypersensitive to Changes in BMP Levels. (A) Frontal view of uninjected neurula (stage 13) stained with telencephalic/eye marker Rx2a, hindbrain marker Krox20, and somite marker MyoD. (B) ADMP-depleted embryos display increased anterior CNS structures (marked by Rx2a) and wider axial structures (enlarged gap between paraxial MyoD expression domains). (C) BMP4-depleted embryos display moderately increased anterior CNS structures (marked by Rx2a). (D) Lowering BMP signaling levels (by injecting Chordin protein in the blastocoele cavity at stage 8) dorsalizes the embryo as shown by increased Rx2a expression, expanded Krox20 expression, and wider axial structures. (E) Lowering BMP signaling levels in ADMP-depleted embryos greatly expands dorso-anterior structures as shown by the dramatic expansion in Rx2a expression and widened axial structures. (F). Lowering BMP signaling levels with Chordin protein in BMP4-depleted embryos fails to expand telencephalic structures marked by Rx2a expression. This difference with the response to ADMP knockdown (compare to panel E) is presumably due to the compensatory up-regulation of ADMP activity on the dorsal side triggered by overall low BMP levels.
	bambi (BMP and activin membrane-bound inhibitor) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 10.5, blastoporal view, dorsal up.

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