Blitz IL et al. (2000), Is chordin a long-range- or short-range-acting ...

XB-ART-10804

Dev Biol 2000 Jul 01;2231:120-38. doi: 10.1006/dbio.2000.9740.

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Is chordin a long-range- or short-range-acting factor? Roles for BMP1-related metalloproteases in chordin and BMP4 autofeedback loop regulation.

Blitz IL , Shimmi O , Wünnenberg-Stapleton K , O'Connor MB , Cho KW .

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Diffusible morphogen models have been used widely to explain regional specification of tissues and body axes during animal development. The three-signal model for patterning the dorsal-ventral axis of the amphibian embryo proposes, in part, that a factor(s) secreted from Spemann's organizer is responsible for converting lateral marginal zone into more dorsal cell fates. We examine the possibility that chordin, a secreted inhibitor of bone morphogenetic protein (BMP) signaling and candidate "dorsalizing signal," is a long-range-acting factor. We show that chordin can, when overexpressed, act directly over distances of at least 450 microm in the early Xenopus embryo to create a gradient of BMP signaling. However, since lower levels of chordin can still induce secondary axes and these amounts of chordin act only locally to inhibit a BMP target gene, we suggest that chordin likely acts as a short-range signal in vivo. Furthermore, BMP1, a secreted metalloprotease that cleaves chordin protein in vitro, inhibits chordin's axis-inducing effects, suggesting that BMP1 functions to negatively regulate chordin's action in vivo. A dominant-negative mutant BMP1 blocks the in vitro cleavage of chordin protein by wild-type BMP1 and induces secondary axes when injected ventrally. We argue that BMP1 and Xolloid are probably functionally redundant metalloproteases and may have two roles in the early Xenopus embryo. One role may be to inhibit the action of low-level chordin protein expressed throughout the entire embryo and a possible second role may be to inhibit activation of a juxtacrine cell relay, thereby confining chordin's action to the organizer region preventing chordin from functioning as a long-range-acting factor.

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Species referenced: Xenopus
Genes referenced: bmp1 bmp2 bmp4 chrd egf gsc h4c4 myc myf5 nog tbx2 tcf3 tll2 ventx2.2

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	FIG. 1. BMP1 is ventralizing. Xenopus BMP1 mRNA was injected radially into the marginal zone of each blastomere of four-cell stage embryos. (A) Stage 43 tadpole following injection of 300 pg BMP1 mRNA/blastomere. Embryos develop ectopic structures often in the ventroposterior region and sometimes in close apposition to the endoderm (data not shown). Embryos injected with lower doses (30–100 pg/blastomere) also develop these structures, but by stage 43 they are no longer morphologically visible (data not shown). (B) Transverse section through the posterior region shown in A (see also inset) shows an abundance of erythrocytes. (C) A transverse section through the heart region (inset) of an uninjected sibling showing erythrocytes for comparison. (D–L) Whole-mount in situ hybridization analyses comparing gene expression from uninjected control embryos (D, G, and J) to wild-type (wt; E, H, and K) and dominant-negative (DN; F, I, and L) BMP1-injected embryos. All embryos are vegetal pole views oriented with dorsal toward the top. Embryos were analyzed at early gastrula stage 10.25–10.5 following microinjection of either 15 pg wtBMP1 or 100 pg DN-BMP1 marginally into each blastomere at the four-cell stage. (D–F) Embryos probed for Xsizzled. (G–I) Embryos probed for Xmyf5. (J–L) Embryos probed for goosecoid.
	FIG. 2. Chordin can create a gradient of BMP signaling. (A) Xvent2 is a direct target of BMP signaling. Animal caps were dissected from late blastula stage 8.5–9 embryos and dissociated into single-cell suspensions. Protein synthesis was inhibited by treatment with cycloheximide (CHX) and the cells were stimulated with purified BMP2 protein at the indicated concentrations. RTPCR analysis for expression of Xvent2 and histone H4 mRNA is shown. 2RT, no reverse transcriptase control. (B) 2357Xvent2/luc differentially responds to varying amounts of BMP signaling. Increasing concentrations of chordin mRNA were co-injected with 20 pg of 2357Xvent2/Luc reporter into a single 64-cell stage blastomere. A semilog plot of luciferase activity at gastrula stage as a function of amount of injected chordin mRNA is shown. (C) Chordin can create a long-range gradient of BMP signaling. Embryos were injected as shown in the inset box. 2357Xvent2/Luc was injected into a single 64-cell stage blastomere, and a second blastomere at the same stage was injected with 1 ng of chordin mRNA. The position of the second blastomere relative to the reporter-injected blastomere is indicated (see inset). “A” indicates the blastomere adjacent to the reporter-injected blastomere. Numbers indicate blastomeres located one, two, or three cells removed from the injected blastomere. Similar experiments were repeated three times. The first two lanes show luciferase activity produced in response to 2 ng of dominant-negative type I BMP receptor or 1 ng of chordin mRNAs co-injected together with the Xvent2 reporter into the same blastomere.
	FIG. 3. Chordin’s range of action is dose dependent. (A–G and J) Chordin mRNA was microinjected together with rhodamine lysinated dextran (RLDx) into a single animal pole blastomere at the 64-cell stage. (D–F and J) The loss of Xvent2 gene expression is shown, as detected by whole-mount in situ hybridization (blue), in a “zone of inhibition” (white) induced by chordin. (A–C and G) Merged images of the bright-field images in D–F and J with corresponding fluorescence images of the RLDx, which marks the progeny of the chordin-injected blastomere. Note that, at higher concentrations of injected chordin, the zone of inhibition of Xvent2 expression expands outside of the RLDx fluorescing “clone” of cells. At the highest dose of chordin shown (300 pg; A, D), the boundary (which is not sharp) between the Xvent2-expressing cells and the zone of inhibition of Xvent2 is approximately 450 mm from the edge of the chordin-expressing fluorescent cell population (yellow arrow). (H, I, K, L) Dominant-negative type I BMP receptor (2 ng) and “cleavage mutant” BMP4 (2 ng) do not act at a distance and behave like low concentrations of chordin in this assay. The asterisk in C marks the narrow area around the chordin-expressing cells which are not expressing Xvent2. (M–O) Xvent2 expression can be rescued by elevating the level of expression of BMP1 in cells within the zone of inhibition of BMP signaling induced by chordin. The arrow in N points to a region of rescued Xvent2 expression overlapping a BMP1-expressing clone of cells (see O). All embryos in A–L, N, andOare animal pole views. (P) Levels of chordin corresponding to chordin’s short-range action are sufficient to induce secondary axes. Tadpole stage embryos showing moderate-sized secondary axes (arrows) induced by injection of 3 pg of chordin mRNA into a single ventral marginal zone blastomere at the 64-cell stage. At this dose 57% of injected embryos (n 5 30) developed secondary axes and 10 pg of chordin induced secondary axes in 75% of injected embryos (n 5 28).
	FIG. 4. Dominant-negative BMP1 inhibits wild-type BMP1’s activity in vitro and in vivo. (A) Construction of a dominant-negative BMP1 protein (DN-BMP1). The C-terminal three CUB repeats and one EGF repeat of BMP1 were fused in frame with the prodomain of Xenopus activinbB, which had its ligand domain discarded. The DN-BMP1 protein is expected to be processed at the activin maturation cleavage site (inverted triangle) upstream of a single FLAG epitope tag located between the cleavage site and the first CUB repeat. (B) Western immunoblotting of transfected COS cell supernatants using anti-FLAG antibody shows that DN-BMP1 is synthesized, secreted, and processed. Lane 1, control untransfected cell supernatant. Lane 2, a band migrating at approximately 60 kDa corresponding to FLAG-tagged processed DN-BMP1. Lane 3, the reduced mobility of DN-BMP1 in lane 2 is likely due to its glycosylation as treatment of transfected cells with the glycosylation inhibitor tunicamycin increases the migration of DN-BMP1 to approximately 52 kDa. (C) DN-BMP1 inhibits chordin proteolysis by wild-type BMP1 in vitro. Transfected 293T cell supernatants were incubated with chordin protein tagged at its C-terminus with a myc epitope (;120 kDa; open arrowhead). Lane 1, control pCS21-transfected supernatants do not contain significant chordin-cleaving activity. Lane 2, supernatant containing wild-type (WT) BMP1 leads to a decrease in the amount of full-length chordin protein and the appearance of faster migrating (;95 kDa; closed arrowhead) cleavage product. Lane 3, supernatant containing DN-BMP1 has no chordin-cleaving activity. Lane 4, DN-BMP1, when cotransfected together with wild-type BMP1, blocks chordin cleavage. (D–H) DN-BMP1 inhibits the activity of wild-type BMP1 and wild-type Xolloid in vivo. (E, G) Expansion of the ventral blood island is observed following microinjection of 15 pg of wild-type BMP1 or wild-type Xolloid marginally into each blastomere at the 4-cell stage (compare to uninjected sibling control in D). (F, H) Injection of 50–150 pg of DN-BMP1 with 15 pg wild-type BMP1 or wild-type Xolloid abrogates the ventral blood island expansion phenotype.
	FIG. 5. Dominant-negative BMP1 is dorsalizing. (A) One hundred picograms of DN-BMP1 was injected into the marginal zone of a ventral vegetal blastomere at the 8-cell stage. Most embryos (89%, n 5 57) develop secondary axes which were incomplete as they lack anterior structures; however, a small percentage (;3.5%) were cyclopic. Different batches of embryos respond differently to DN-BMP1 (data not shown). In many batches, 100 pg is insufficient to induce secondary axes, but 300 pg will induce secondary axes efficiently. Usually, 1 ng of DN-BMP1 induces gastrulation defects. At these higher doses of DN-BMP1, the appearance of cyclopic heads and heads containing two eyes rises to approximately 30% of injected embryos. (B) Co-injection of 30 pg lacZ mRNA together with 100 pg DN-BMP1 into a single ventral vegetal blastomere at the 8-cell stage results in strong staining in the secondary axes of tailbud stage embryos. This result shows that DN-BMP1 functions in the region where it is expressed, converting ventral marginal zone to dorsal organizer tissue, and does not need to act at a distance to induce secondary axes. (C) Microinjection of 100 pg of DN-BMP1 into the marginal zone of both ventral blastomeres at the 4-cell stage induces secondary axes (arrows), and formation of these axes can be blocked (D) by co-injection with 60–100 pg of wild-type BMP1 or Xolloid mRNA (data not shown).
	FIG. 6. Axis rescue by dominant-negative BMP1 suggests that chordin expressed outside of Spemann’s organizer may be negatively regulated by BMP1 and Xolloid. (A) Hypothesis 1: Chordin protein, synthesized by cells in Spemann’s organizer (left, dark blue), may act at a distance (e.g., by diffusing away from the organizer; arrows). Normally, this chordin protein may be the target of degradation by endogenous BMP1 and Xolloid. DN-BMP1 blocks chordin’s degradation leading to development of secondary axes. (B) Hypothesis 2: Chordin protein, while synthesized by cells in Spemann’s organizer (left, dark blue), may also be distributed at a low level throughout the embryo (left, light blue), perhaps as maternally expressed mRNA or protein or as low-level uniform zygotic expression. DN-BMP1 expression on the ventral side may block BMP1 and Xolloid thereby allowing this uniformly expressed chordin protein to induce development of secondary axes. Embryos depicted to the right in A and B show the effects of UV treatment during the first cell cycle, which blocks expression of chordin in the organizer at gastrula stage (Sasai et al., 1994, and data not shown), not nonorganizer chordin expression. Therefore, assessing DN-BMP19s ability to induce an axis in UV-hyperventralized embryos permits determination of whether organizer expression of chordin is necessary for the axis-inducing activity of DN-BMP1 (C–F). (C) UV-irradiated embryos which are hyperventralized (DAI 5 0.13, n 5 46). (D) UV-treated embryos injected marginally at the 8-cell stage with 100 pg of DN-BMP1 mRNA (DAI 5 3, n 5 27). (E) Axis rescue was blocked by co-injection of either 60 pg wild-type (WT) BMP1 or 60 pg wild-type Xolloid (data not shown) mRNAs together with 100 pg DN-BMP1. Therefore, we conclude that DN-BMP1 functions to induce axes by blocking endogenous BMP1 and Xolloid. (F) Sibling control embryos used for staging. (GL) Axis rescue by DN-BMP1 in embryos hyperventralized by dominant-negative TCF3 (DN-TCF3). It has previously been shown (Laurent et al., 1997; Medina et al., 1997) that inhibition of organizer formation by UV treatment during the first cell cycle results in ectopic expression of some organizer marker genes in the vegetal pole. We wished to rule out the possibility that axis rescue by DN-BMP1 could be due to a modified version of hypothesis 1, in which chordin is instead expressed in the vegetal pole and “diffuses” into the marginal zone. Similar to hypothesis 1 above, degradation of this chordin by BMP1 and Xolloid may be inhibited by DN-BMP1. To rule out this possibility, we sought to rescue axis formation using DN-BMP1 following inhibition of organizer formation by DN-TCF3. (G) Hyperventralized embryos following injection of 250 pg marginally into the dorsal side of each blastomere at the 2-cell stage. lacZ mRNA was co-injected together with DN-TCF3 to mark tissue derived from the injected side. (H) Axes in embryos injected dorsally with DN-TCF3 and lacZ were partially rescued by ventral injection at the 8-cell stage with 100 pg of DN-BMP1 into each of the two vegetal blastomeres. Note that the lacZ staining, marking cells which would have been dorsal, now marks ventral cells, while axes are derived from unmarked ventral tissue. (I) Control embryos injected in the dorsal marginal zone with lacZ mRNA alone showing the normal distribution of lacZ into dorsal structures. (J) Control embryos injected in the ventral marginal zone with lacZ mRNA alone showing the normal distribution of lacZ into ventroposterior structures. (K) DN-TCF3 inhibits chordin expression in the organizer. (L) A sibling embryo showing chordin expression in the organizer. K and L are vegetal views. Dorsal is toward the top in L.
	FIG. 7. Chordin is expressed outside of Spemann’s organizer and BMP1 acts to inhibit a chordin positive autofeedback loop. (A) Chordin is expressed outside of Spemann’s organizer. Marginal zones fragments were dissected from early gastrula stage 10.25 embryos (numbers on the embryo correspond to RT-PCR lane numbers) and chordin RNA was examined by RT-PCR. While chordin is expressed at very high levels dorsally, it is detectable in the ventral marginal zone as well. (B–E) Chordin gene expression is up-regulated by repressing BMP signaling. Ventral expression of chordin itself (lacking its 39UTR; 20 pg), dominant-negative type I BMP receptor (1.6 ng), or noggin (10 pg) results in ventral expression of chordin mRNA. (E) Chordin is also induced in the animal pole in response to inhibition of BMP signaling (arrow). Noggin mRNA (10 pg) was co-injected animally at the 64-cell stage. (F) Chordin is also up-regulated ventrally and in the animal pole (G) upon expression of DN-BMP1. In B–G, the mRNA injected is stated in the lower right corner. In B, chordin’s 39UTR was used as probe to selectively detect mRNA synthesized off the endogenous chordin gene.
	FIG. 8. Competition between BMP4 and chordin positive autofeedback loops. BMP4 signaling (left) through heteromeric type I and II BMP receptor complexes results in the concomitant repression of chordin gene expression and activation of BMP4 gene expression, thereby maintaining expression of BMP4 outside the Spemann’s organizer. A chordin positive autofeedback loop (right) occurs indirectly through chordin’s repression of the BMP4 positive autofeedback loop. Chordin, by competitively inhibiting BMP4 receptor activation, blocks BMP4 signaling-dependent activation of its own gene expression and, concomitantly, chordin blocks BMP49s repression of expression of the chordin gene. The chordin gene is then derepressed, completing the loop. The balance between these two feedback pathways depends on the levels of expression of BMP4 (ventrally, laterally, and animally) and chordin (dorsally; Spemann’s organizer). These are established at midblastula transition by the dorsal determinant pathway, in the case of chordin, and the “default” ventral state of the remainder of the embryo, also established by maternal factors. Low level expression of chordin protein outside of the organizer is inhibited by BMP1 and Xolloid which proteolytically inactivate chordin, thereby permitting maintenance of the ventral (BMP4 autofeedback) pathway. BMP signal transduction resulting in activation and repression of the BMP4 and chordin genes may or may not occur by direct pathways.