Ambrosio AL , Taelman VF , Lee HX , Metzinger CA , Coffinier C , De Robertis EM .

HD21502-22 NICHD NIH HHS , R01 HD021502-22 NICHD NIH HHS , R01 HD021502 NICHD NIH HHS , R37 HD021502 NICHD NIH HHS

	Figure 1. CV2 Is a Secreted BMP Feedback Inhibitor(A) Normal CV2 expression at stage 22 (hemisection). vb, ventral blastopore.(B) CV2MO microinjection increases CV2 expression.(C) The CV2 negative feedback loop requires BMP4. For each experimental sample, at least 25 embryos were examined, with similar results.(D) qRT-PCR showing increased expression of the ventral marker Vent1 in CV2-depleted embryos.(E) CV2 depletion reduces expression of the dorsal/forebrain marker Six3.(F) Endogenous Smad1 phosphorylation is increased by CV2 depletion at gastrula and neurula stages 12, 13, and 14. A pSmad1 signal was detectable in the stage 14 uninjected lane upon longer exposure. Total Smad1 antibody (T-Smad1) staining was used as loading control.
	Figure 2. CV2 and Chordin Compensate for Each Other in Xenopus D-V Patterning(A) Uninjected embryo showing CV2 expression, which is used here as a BMP4 signaling readout (n = 17). Inset shows mid-gastrula embryo stained for Chordin (Chd) and Sizzled (Szl) (n = 24).(B) CV2 depletion upregulates its own expression (n = 15), as well as increasing Szl and decreasing Chd (n = 20).(C) Depletion of the BMP antagonist Chordin also increases the ventral CV2 and Szl expression domains (n = 13 and n = 18, respectively).(D) When coinjected, CV2 MO and Chd MO show a marked expansion of the CV2 and Szl expression domains (n = 15 and n = 25, respectively).(E–H) qRT-PCR analyses of single and double CV2 and Chd morphants for the D-V markers Szl, CV2, Gsc, and Chd at late gastrula stage 12.5.(I) Endogenous Smad1 phosphorylation is increased by coinjection of Chd MO and CV2 MO in stage 11 embryos.(J) The CV2 cleavage sequence contains the conserved low-pH GDPH autocatalytic site present in mucins. hMuc2, human mucin-2.(K) Chordin, but not full-length CV2, is cleaved by the extracellular zinc-metalloproteinases Xolloid-related (Xlr) and BMP1 (lanes 1–6). However, cleavage of full-length CV2 (80 kD band) is triggered by low pH (lanes 9 and 10).(L–N) Ventral injections of mRNAs encoding full-length CV2 (CV2-FL, n = 56, of which 95% had partial secondary axes, three independent experiments), N-terminal CV2 fragment terminating at the GDPH cleavage site (CV2 N-Ter, n = 42, no secondary axes observed), or a secreted C-terminal fragment encoding most of the vWFd domain (CV2 C-Ter, n = 45, no secondary axes observed). The insets show injected embryos at late neurula stage hybridized with the panneural marker Sox2.
	Figure 3. Twisted-Gastrulation Is Required for the Effects of CV2 Loss of Function and Overexpression(A) Simultaneous depletion of CV2 and Chordin strikingly increased CV2 expression, reflecting increased BMP signaling (see Figure S5 for controls).(B) The effects of CV2 MO and Chd MO require Twisted-Gastrulation (Tsg) activity (pro-BMP effect of Tsg).(C) CV2 protein injection into the blastula cavity induces strong dorsalization of the Xenopus embryo, as indicated by the expansion of the dorsal markers Xag1, Six3, and Krox20 (n = 12, all strongly dorsalized). Inset shows an uninjected embryo.(D) CV2 protein requires endogenous Tsg for its anti-BMP activity (n = 10, all embryos similarly affected). Inset shows Tsg MO-injected embryo.(E) CV2 protein injection expands the neural tube (n = 17). Inset shows uninjected embryo.(F) Tsg and CV2 protein coinjection renders CV2 a stronger BMP antagonist, expanding the nervous system marked by Sox3 (n = 16, all coinjected embryos were more dorsalized than those injected with CV2 protein alone despite some individual variations). Inset shows embryo injected with Tsg protein alone.
	Figure 6. The Pro-BMP Function of CV2 Is Revealed in Epistatic Experiments with Chordin or Tolloid(A) Expression of the eye field marker Rx2a in uninjected Xenopus late neurula embryo (n = 45), anterior view.(B) Chordin protein injection (2 μM, 60 nl) into the blastocoele at late blastula (stage 9.5) caused dorsalization and an increase in Rx2a expression (n = 54).(C) CV2-depleted hosts were more sensitive to the anti-BMP effects of Chordin, as indicated by the expansion in the Rx2a domain (n = 48).(D) Uninjected early neurula (stage 13, side view) showing Otx2 expression in the future forebrain and midbrain regions (n = 19).(E) Chordin protein injection expands Otx2 in wild-type embryos (n = 25).(F) CV2 depletion sensitizes the embryo to the effects of Chordin on Otx2 (n = 23). Note that the border of Otx2 expression expands posteriorly.(G–I) qRT-PCR analysis of the D-V markers Chd, CV2, and Szl after Chordin protein injection into wild-type and CV2-depleted embryos at late blastula. The bars indicate standard deviation between two groups of seven embryos each.(J and K) Anterior views of uninjected control or embryo microinjected four times with 250 pg of DN-Xlr mRNA, which inhibits the proteolytic degradation of Chordin. Note that the Otx2-positive forebrain (fb), midbrain, and cement gland (cg) regions are expanded, consistent with the anti-BMP effects of Tolloid inhibition (n = 27).(L) In CV2-depleted embryos, Otx2 expression is greatly expanded by DN-Xlr mRNA (n = 27). The dotted line indicates the eye field (eye), which is more weakly stained by Otx2.
	Figure S1. CV2 mRNA Is Expressed in Regions of High BMP and Its Depletion Ventralizes Xenopus Embryos. The expression pattern of CV2 in Xenopus embryos has not been previously reported. CV2 was expressed in all three embryonic layers, ectoderm, mesoderm, and endoderm in domains overlapping with those of BMP4 expression [Fainsod, A, Steinbeisser, H., and De Robertis, E.M. (1994). EMBO J. 13, 5015-5025]. At later stages, CV2 expression was detected in other regions of high BMP signaling such as the dorsal region of the eye and the cement gland, the roof plate, the hypochord, and blood islands. Before gastrulation, CV2 transcripts were undetectable by in situ hybridization or RT-PCR. (A) In a hemisected mid-gastrula stage embryo, CV2 is expressed in broadly in ventral regions. Bracket marks the blastopore. (B) At late gastrula, CV2 expression concentrates around the ventral blastopore. (C) In a neurula stage hemisected embryo, CV2 expression is seen in the ventral endoderm around the blastopore marked by bracket. (D) CV2 expression is turned on in the dorsal eye, the cement gland and is still present in the ventral endoderm in hemisected tadpoles. (E) CV2 is expressed in high BMP regions both in the ventral and the dorsal side of transversely sectioned embryos. Inset shows CV2 expressed in the roof plate of the spinal cord and in the hypochord. (F) RT-PCR analysis indicates CV2 expression is zygotic, starts at gastrula, and reaches its maximum at neurula stage. (G) Stage 32 uninjected embryo (inset shows stage 40). (H) The CV2MO injected embryo shows a reduced head and dorsal (white arrow) and ventral fins and a strikingly smaller eye (inset). (I) Uninjected embryo stained with eye marker Six3 and the ventral BMP signaling marker Sizzled. (J) CV2 depletion reduces the eye marker Six3 and increases ventral BMP signaling marked by Sizzled. bc, blastocoele; vb, ventral blastopore; ve, ventral endoderm; ar, archenteron; ey, eye; cg, cement gland; bi, blood island; rp, roof plate; sc, spinal cord; no, notochord; hy, hypochord
	Figure S2. Maternal Injection of CV2 MO into Xenopus tropicalis Oocytes (A) Uninjected embryo at stage 33. (B) The CV2 MO oocyte injection phenotype does not differ from the one obtained when embryos are injected at 2-cell stage. Note the reduction in the ventral fin (vf), which indicates a pro-BMP function for endogenous CV2. This experiment indicates that there is no significant maternal contribution to the CV2 phenotype in Xenopus.
	Figure S3. CV2 Knockdown Affects D-V Patterning in the Mesoderm and the Endoderm. (A-B) Embryos stained with the blood island marker Scl (Stem cell leukemia). CV2 depletion increases staining in the blood island indicating an increase in BMP signaling. (C-D) In situ hybridization using the endodermal marker Cad2. CV2 depletion severely affects endoderm formation. Note that the ventral fin (vf) is decreased in size.
	Figure S4. The CV2 MO Is Effective and Can Be Rescued by mCV2 mRNA. (A) CV2 MO inhibits the translation of microinjected Xenopus CV2 mRNA. Embryos were injected four times with 250 pg of mRNA with or without CV2 MO, harvested at stage 13 and analyzed by Western blot using α-tubulin (1:2000, Calbiochem) as loading control. The novel anti-xCV2 antibody used here (1:2000 dilution) was generated in rabbit (Covance) from a synthetic peptide encoding the amino-terminal sequence of Xenopus CV2 (SSFLTGSIAKAENEGEALQIPFITDNPC). (B) Hemisected embryo stained with CV2. (C) Expansion of the ventral CV2 domain after CV2 MO radial injection. (D) Mouse CV2 mRNA (Coffinier et al., 2002) prevents the effect of CV2 MO. The white brackets indicate the width of the CV2 domain around the ventral blastopore.
	Figure S5. Tsg Functions as a Potent Pro-BMP Agent in the Absence of CV2 and/or Chordin. (A) In situ hybridization of hemisected embryo stained with CV2; uninjected stage 22 embryo. CV2 transcripts are used in this experiment as a reporter for BMP activity. (B) Upregulation of CV2 expression after CV2 MO injection. (C) Chordin depletion results in an expansion of CV2 expression domain. (D) A cooperative effect is observed after co-injection of CV2 MO and Chd MO. (E) Depletion of Tsg alone resembles wild-type embryos or slightly decreases BMP signaling. The same Tsg oligomer as in Blitz et al. (2003) was used in these experiments. (F) Tsg depletion completely erased the expression of CV2 in CV2 MO injected embryos. (G) Tsg knockdown reduced the CV2 expression domain in the Chd MO injected embryos. (H) Tsg is required (pro-BMP effect) for the striking increase in the expression of CV2 observed in CV2 MO and Chd MO injected embryos. (I-L) Effects of Tsg MO alone using Sizzled as the BMP signaling readout. Injection of Tsg MO into each cell at 4-cell stage consistently decreased Szl (n=25) when compared to uninjected stage 19 embryos (n=23). This is in agreement with findings in zebrafish with Tsg MO (Little and Mullins, 2004; Xie and Fisher, 2005). Microinjection of Tsg MO into the two dorsal blastomeres of embryos with clear D-V polarity inhibited dorsal Szl but increased its expression on the ventral side (n=24). This phenotype is probably caused by the inhibition of Chd protein function, which requires Tsg, and is consistent with phenotypes reported by Blitz et al. (2003). The depletion of Tsg in the two ventral blastomeres decreased Szl expression on the ventro- posterior region, but did not affect Szl expression in the anterior domain, which derives from non-injected cells (n=21). Differences in the phenotypes of dorsal and ventral injections of Tsg MO in Xenopus have been reported previously (Blitz et al., 2003). We propose that these D-V differences can be explained if dorsal depletion of Tsg caused predominantly defects in the function of Chordin, and ventral depletion reflected predominantly the requirement of Tsg for proper CV2 and BMP function. The most striking effect in this figure is seen by comparing the embryos in panels D and H, which are reproduced in the main text (Figure 3A and 3B). When both CV2 and Chordin are depleted, BMP signaling strongly requires Tsg. It is known that Tsg binds to BMP on its own right (Oelgeschläger et al., 2000) and that its presence does not affect significantly the binding of BMP to its receptors (Larraín et al., 2001). Tsg may have a permissive function in BMP signaling, perhaps helping maintain BMP ligands in a soluble state in the extracellular space.
	bmper (BMP binding endothelial regulator) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, vegetal/blastoporal view, dorsal up. Key: vb= ventral blastopre region
	bmper (BMP binding endothelial regulator) gene expression in a hemisected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17-18, anterior left, dorsal up. Key: vb= ventral/lower blastopre lip, ve= ventral endoderm, bc= blastocoel; ar= archenteron.
	bmper (BMP binding endothelial regulator) gene expression in a hemisected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, anterior left, dorsal up. Key: cg= cement bland; en= endoderm; vb= ventral blastopore region
	bmper (BMP binding endothelial regulator) gene expression in a transverse section of a Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, dorsal up. Key: ar: archenteron; ve= ventral endoderm; bi= ventral blood island; rp= roof plate, no= botochord; hy= hypochord; sc= spinal cord.
	Figure 7Model of the Molecular Interaction of CV2, Chordin, Tsg, and BMP4 (A) Model of the regulation of D-V patterning by a network of extracellular proteins secreted by the dorsal and ventral centers of the Xenopus gastrula. Arrows in black indicate direct protein-protein interactions in the extracellular space, blue arrows transcriptional regulation by the BMP-responsive transcription factors Smad1/5/8, and the red arrow the hypothetical flux of Chordin/ADMP/BMP from the dorsal toward the ventral center of the embryo, where it would bind to CV2. This model of D-V patterning is self-regulating because at low BMP levels the transcription of the BMP-like molecule ADMP is activated, and at high BMP levels the BMP antagonist CV2 and the tolloid inhibitor Sizzled are upregulated (Reversade and De Robertis, 2005, Lee et al., 2006). The function of Tsg is to both increase BMP inhibition by CV2 and Chd and to promote BMP4 signaling in their absence. The tolloid protease Xlr cleaves Chordin/ADMP/BMP complexes, releasing active BMPs concentrated on the ventral side. (B) Model in which Chordin flow would help transport BMPs and Chordin from the dorsal to the ventral side of the Xenopus embryo. Three possible outcomes are indicated.

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