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In Xenopus, dorsal-ventral (D-V) patterning can self-regulate after embryo bisection. This is mediated by an extracellular network of proteins secreted by the dorsal and ventral centers of the gastrula. Different proteins of similar activity can be secreted at these two poles, but under opposite transcriptional control. Here we show that Crescent, a dorsal protein, can compensate for the loss of Sizzled, a ventral protein. Crescent is a secreted Frizzled-Related Protein (sFRP) known to regulate Wnt8 and Wnt11 activity. We now find that Crescent also regulates the BMP pathway. Crescent expression was increased by the BMP antagonist Chordin and repressed by BMP4, while the opposite was true for Sizzled. Crescent knock-down increased the expression of BMP target genes, and synergized with Sizzled morpholinos. Thus, Crescent loss-of-function is compensated by increased expression of its ventral counterpart Sizzled. Crescent overexpression dorsalized whole embryos but not ventral half-embryos, indicating that Crescent requires a dorsal component to exert its anti-BMP activity. Crescent protein lost its dorsalizing activity in Chordin-depleted embryos. When co-injected, Crescent and Chordin proteins greatly synergized in the dorsalization of Xenopus embryos. The molecular mechanism of these phenotypes is explained by the ability of Crescent to inhibit Tolloid metalloproteinases, which normally degrade Chordin. Enzyme kinetic studies showed that Crescent was a competitive inhibitor of Tolloid activity, which bound to Tolloid/BMP1 with a K(D) of 11 nM. In sum, Crescent is a new component of the D-V pathway, which functions as the dorsal counterpart of Sizzled, through the regulation of chordinases of the Tolloid family.
Fig.1. Xenopus crescent is expressed dorsally and repressed by BMP signaling. (A) D–V Patterning is regulated by proteins secreted by the dorsal and ventral signaling centers. For the proteins listed, proteins of similar function are secreted by the two sides, but under opposite transcriptional control. (B) sFRPs of Xenopus (x), human (h), zebrafish (z) and chicken (ch) origin were compared using Molecular Evolutionary Genetics Analysis (MEGA) software (Tamura et al., 2007). Crescent and Sizzled are philogenetically related, and distant from the other sFRPs. (C–E) Crescent expression is under negative transcriptional control by BMP4 signaling. Microinjection of Chordin (Chd) protein increases crescent transcripts, while microinjection of BMP4 protein decreases crescent expression in stage 12 gastrulae. (F-H) sizzled expression is inhibited by injection of crescent mRNA and markedly expanded upon depletion of Crescent (Cres MO); an uninjected sibling at stage 11 is shown for comparison.
Fig.2. Crescent regulates BMP signaling. (A,B) Microinjection of crescent mRNA expands the expression of chordin, a gene that is negatively regulated by BMP signaling. (C) Diagram showing a control crescent mRNA containing wobble position mutations is no longer targeted by Crescent MO. (D–G) Crescent MO greatly expands expression of the ventral (high-BMP) marker sizzled, while crescentWobble mRNA reduces Sizzled expression and rescues the effects of Crescent MO. (H–K) Knockdown of Crescent increases the transcript levels of BMP induced genes (Xlr, BMP4, Vent-1), while reducing the levels of chordin, a gene repressed by BMP signaling. (L–O) chordin mRNA expression is reduced by Crescent MO or Sizzled MO. Note that simultaneous depletion of both sFRPs causes a synergistic ventralizing effect, greatly reducing chordin transcripts at early gastrula. (P) Model in which the BMP gradient is represented by a see-saw in which dorsal and ventral inhibitors of Tolloid metalloproteinases adjust the D–V gradient through the proteolytic degradation of Chordin. Blue arrows symbolize transcriptional regulation by BMPs, black arrows indicate direct protein–protein interactions.
Fig.5. Crescent requires Chordin in order to dorsalize the embryo. (A) Embryos bisected along their D–V axis. The dorsal half self-regulates, forming a well-proportioned embryo, while the ventral half forms a belly piece consisting of ventral tissues. (B) Crescent mRNA microinjection increases SOX2 expression in dorsal halves, but has no effect on ventral half-embryos. Thus, the dorsalizing activity of Crescent requires a dorsal component. (C) Uninjected control embryos showing normal Rx2a and Sizzled transcript levels at stage 20. (D) Embryos injected with Chordin MO showing a ventralized phenotype consisting of reduced Rx2a and expanded posteriorSizzled transcripts (inset). (E) Injection of Crescent protein into the blastocoele dorsalizes embryos, expanding Rx2a expression, decreasing Sizzled expression in the posterior-ventral region and increasing Sizzled in the anterior-ventral region (where BMP2 is expressed). (F) Injections of Crescent protein into Chordin-depleted embryos are without dorsalizing effects; this result indicates that Crescent protein requires Chordin to dorsalize Xenopus embryos. Insets show lateral views.
Fig.6. A Crescent mutant mimicking the Ogon mutation lacks Tolloid inhibitory activity and has less anti-BMP activity in the Xenopus embryo. (A) Flag-tagged affinity-purified protein CrescentWT and CrescentD103N. (B–D) Microinjection of CrescentWT protein (2.5 μM) into the blastocele dorsalizes embryos and expands Rx2a expression, while microinjection of the same concentration of CrescentD103N had reduced dorsalizing ability and was unable to expand Rx2a expression. (E, F) CrescentWT inhibited cleavage of a fluorogenic Chordin peptide by BMP1 enzyme in a dose-dependent manner, whereas CrescentD103N was unable to inhibit this reaction. (G–I) CrescentD103N is able to inhibit the induction of secondary axes by xWnt8 mRNA, indicating that the Wnt-inhibiting and Tolloid-inhibiting activities of CrescentWT are separable. (J–L) A sub-threshold amount of Chordin protein injected into the blastocoele has very limited effect. If, in addition to this amount of Chordin, embryos also received a modest amount of CrescentWT, synergetic cooperation between Crescent and Chordin proteins was observed, manifested as an extreme increase in Rx2a expression in ectoderm. Embryos injected with CrescentD103N, although retaining a dorsally “kinked†phenotype (probably caused by inhibition of convergence and extension movements that require Wnt signaling), did not exhibit this increase in the Rx2aforebrain marker, when co-injected with Chordin protein. Insets show frontal views of embryos without injection of Chordin protein.
Fig.8. The authors thank Jack Greenan and D. Geissert for technical assistance, members of our laboratory for discussions and comments on the manuscript, and Drs. R. Lehrer and Grace Jung for invaluable help with the BIAcore analyses. Doctoral studies by D.P. are supported by a Fulbright Science and Technology Award. This work was supported by the NIH (HD21502-24). E.M.D.R. is a Howard Hughes Medical Institute investigator.
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