Piccolo S , Agius E , Lu B , Goodman S , Dale L , De Robertis EM .

R37 HD21502-11 NICHD NIH HHS , R37 HD021502-13 NICHD NIH HHS , R37 HD021502 NICHD NIH HHS

	Figure 1. Xolloid (XLD) Ventralizes Ectoderm and Mesoderm (A–H) Ventralization of ectoderm and mesoderm revealed by whole-mount in situ hybridization. Uninjected (left) and XLD mRNA–injected embryos (right) were probed for the indicated markers. Injections were done radially with 200 ng of XLD mRNA per blastomere at the 8-cell stage in the animal cap (A and B) or in the marginal zone (C–H). (A-D) Neurula stage embryos stained for (A) the neural marker Sox-2, (B) the neural crest marker slug, (C) the dorsal mesoderm and floor plate marker Shh, and (D) the dorsal mesoderm marker MyoD. Note the reduction of the neural and dorsal mesodermal fields. (E) At midgastrula stages, the ventrolateral marker Xwnt8 is expanded and expressed in the organizer in Xld-injected embryos. (F) At neurula stages, the BMP-4 domain is expanded in Xld-injected embryos, reducing the size of the neural plate. (G and H) Chd at early and late gastrula stages, respectively. Note that the transcription of Chd in Xld-injected embryos starts normally but collapses during the course of gastrulation. (I) Normal (top) and XLD-injected 3-day tadpoles (bottom). Embryos were injected radially with 200 pg of XLD mRNA in each blastomere at the 4-cell stage. Note the recovery to a relatively mild ventralization (93% affected, DAI = 3.2, n = 311) when compared with the severe reduction in axial and neural markers observed at the early stages.
	Figure 2. Xolloid Acts Upstream of BMP Receptor and Blocks Secondary Axes Induced by chordin but Not by noggin or follistatin (A) Double axes and dorsalized embryos induced by injection of 150 pg of DNBMPR mRNA (67%, n = 57). (B) Double axes and dorsalized embryos induced by coinjection of 150 pg of DNBMPR with 400 pg of XLD mRNA (62%, n = 53). (C) Secondary axes induced by injection of 100 pg of chd (69%, n = 71). (D) XLD mRNA (400 pg) negates the activity of 100 pg of CHD mRNA (2%, n = 128). (E) Coinjection of 20 pg of noggin mRNA with 400 pg of XLD mRNA results in secondary axis induction (57%, n = 75) at a similar frequency as 20 pg of noggin mRNA alone (48%, n = 55, data not shown). (F) Coinjection of 150 pg of follistatin mRNA with 400 pg of XLD mRNA results in secondary axis induction (61%, n = 32) at a similar frequency as 150 pg of follistatin mRNA alone (64%, n = 40, data not shown). All embryos were injected in a ventral blastomere at the 4-cell stage. Note that Xolloid specifically blocks the activity of chordin.
	Figure 3. Xolloid Is a Secreted Protease That Cleaves Chordin Protein at Two Specific Sites (A) Xolloid is secreted. Recombinant XLD was detected by an anti-Flag monoclonal antibody that recognizes an epitope tag placed at the COOH termini of XLD and of DN-XLD. Lane 1, control conditioned medium (c.m.) from 293T cells transfected with vector DNA only; lanes 2 and 3, equivalent aliquots of conditioned medium from Xld and DN-Xld transfections, respectively. Note that processed Xolloid protein of approximately 130 kDa is secreted into the medium; the faint band at 150 kDa corresponds to the proenzyme. (B) Xolloid cleaves Chordin. Western blot analysis of CHD protein probed with an anti-Myc antibody that recognizes its COOH terminus. CHD protein was incubated for 10 hr as described in the Experimental Procedures with the following solutions: lane 1, control 293T conditioned medium is devoid of cleaving activity; lane 2, Xolloid-containing medium specifically cleaves CHD, removing an NH2-terminal fragment; lane 3, Xolloid is inactivated by incubation with 1–10 ortophenanthroline, an inhibitor of zinc metalloproteases; lane 4, Xolloid retains enzyme activity in the presence of a protease inhibitor cocktail that does not affect metalloproteases but should block serine, aspartic, and cysteine proteases (see Experimental Procedures for details); lane 5, the DN-Xld point mutation is catalytically inactive; lane 6, CHD-Myc protein used as substrate for purified enzyme; lane 7, affinity-purified Xolloid-Flag protein is able to cleave CHD. (C–E) Mapping the Xolloid cleavage sites on Chordin. Chordin fragments were visualized by (C) anti-NH2-CHD peptide, by (D) anti-COOH-Myc, and by (E) anti-Internal-CHD (αI) antibodies. The resulting proteolytic fragments are indicated. (C–E) Lane 1, CHD is full-length after incubation with control medium. Lane 2, Xolloid cleaves CHD at two sites. Lane 3, before addition of Xolloid, CHD was preincubated for 2 hr at 25°C with a 5 molar excess of recombinant human BMP-4, to cause the formation of CHD/BMP-4 complexes. (F) Noggin protein is not cleaved by Xolloid. Lanes 1, 2, and 3 are Noggin in control medium, Noggin in Xolloid medium, and Xolloid medium with Noggin-BMP-4 complexes, respectively. Acrylamide percentages in the SDS–PAGE gels were as follow: (A) and (B) 7%; (C) 6%–18% gradient; (D–F) 7.5%–18% gradient. For samples in gel (E), the incubation time was extended to 16 hr.
	Figure 4. Cleavage of Chordin Causes Loss of Dorsalizing and BMP Blocking Activity (A–C) External views of ventral marginal zones (VMZs): (A) treated with full-length CHD protein in control medium, (B) treated with XLD-cleaved Chordin protein, and (C) untreated VMZs. Note the lack of elongation in (B). (D) Dorsal mesoderm is not induced in VMZs treated with digested CHD. RT–PCR analysis was used to score for expression of the muscle-specific marker α-actin in VMZs (n = 20 per lane). EF1α (elongation factor 1α) provides a loading control. Note that digestion with Xolloid inactivates dorsalization by Chordin. (E) Cleaved CHD is unable to block the activity of BMP-4/7. Animal caps were dissected at blastula stage 8 and incubated with the indicated proteins until siblings reached stage 10̂. Lane 1, untreated animal caps (AC). Lane 2, incubation in 0.7 nM BMP-4/7 induces the ventral mesodermal marker Xhox3. Lanes 3–5, animal caps plus 0.7 nM BMP-4/7 preincubated for 2 hr at 25°C with, respectively, CHD, CHD cleaved by XLD, and CHD incubated in control medium. Note that Chordin preincubated with Xolloid is unable to block BMP signaling.
	Figure 5. Xolloid Digestion Causes the Release of Biologically Active BMP-4/7 from Inactive CHD/BMP Complexes Top: experimental design. Bottom: RT–PCR of dissociated-reaggregated animal cap cells. N-CAM is used as a pan neural marker, keratin as an epidermal-specific marker, XBRA as a mesodermal marker, and EF1α as a loading control. Lane 1, stage 18 whole embryos. Lanes 2 and 3, autoneuralization, denoted by the high NCAM and low keratin band intensities, caused by cell dispersal in, respectively, uninjected and Xld-injected animal caps. Lane 4, BMP-4/7 protein (0.7 nM) induces epidermis and suppresses neural induction. Lane 5, BMP signaling is blocked by preincubation of BMP-4/7 with Chordin (1 nM). Lane 6, injection of XLD mRNA into animal cap cells at the 8-cell stage causes release of active BMP-4/7 from the previously inactive complex (compare to lane 5).
	Figure 6. In Vivo Role of Xolloid: Expansion of Organizer Activity in Embryos Injected with Dominant-Negative Xolloid (A) Top, schematic structure of Xolloid: EGF and C1r/s are protein–protein interaction domains. Bottom, sequence of the protease domain of Drosophila Tolloid and of Xenopus Xolloid surrounding a conserved tyrosine (circled, position 272 in Tolloid and 296 in Xolloid) mutated in DN-Xld. (B) Control stage 29 embryo. (C) Xld gain-of-function: ventralized embryo injected with 200 pg of XLD mRNA in each blastomere at the 4-cell stage. (D) Dorsalization in embryos similarly injected with 300 pg of DN-XLD mRNA (76%, n = 127). Compare with sibling embryo in (B); the head and cement gland are enlarged, reflecting dorsalization of the embryos. (E–G) In situ hybridization for the indicated markers in control (left) or DN-XLD mRNA–injected (right) embryos. The neural domain marked by Sox-2 (E) and the dorsal mesoderm domains marked by Chd (F) or MyoD (G) are expanded and increased in intensity in DN-Xld-injected embryos (n = 24, 10, and 15, respectively), indicating increased organizer activity. The injected embryo in (E) is shortened due to dorsalization. (H) Stage 20 embryos injected in the two dorsal blastomeres at the 8-cell stage with 200 pg of Xolloid mRNA. (I) Normal development is restored by coexpression of XLD mRNA (200 pg) and DN-XLD mRNA (800 pg). This indicates that DN-Xld can antagonize wild-type Xld. (J) Western blot showing that DN-Xld protein produced by cotransfection inhibits the enzymatic activity of Xolloid on Chordin substrate.

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