LAUSR

Significance The early frog embryo provides a classical model system for the isolation of secreted molecules that regulate long-range cell–cell communication. Extensive screens of a region with embryonic induction activity, called Spemann organizer, have revealed a large number of secreted growth factor antagonists. Here, we used high-throughput sequencing of differentiating ectodermal explants to isolate yet another potent Wnt inhibitor expressed in Spemann organizer tissue. Bighead is a secreted protein that inhibits Wnt by causing the endocytosis and degradation in lysosomes of the Wnt coreceptor Lrp6. Its overexpression induces embryos with larger heads, and its knockdown reduces head development through the regulation of Wnt signaling. Many Wnt inhibitors exist, and we find that endocytosis regulation is crucial for function. The Xenopus laevis embryo has been subjected to almost saturating screens for molecules specifically expressed in dorsal Spemann organizer tissue. In this study, we performed high-throughput RNA sequencing of ectodermal explants, called animal caps, which normally give rise to epidermis. We analyzed dissociated animal cap cells that, through sustained activation of MAPK, differentiate into neural tissue. We also microinjected mRNAs for Cerberus, Chordin, FGF8, BMP4, Wnt8, and Xnr2, which induce neural or other germ layer differentiations. The searchable database provided here represents a valuable resource for the early vertebrate cell differentiation. These analyses resulted in the identification of a gene present in frog and fish, which we call Bighead. Surprisingly, at gastrula, it was expressed in the Spemann organizer and endoderm, rather than in ectoderm as we expected. Despite the plethora of genes already mined from Spemann organizer tissue, Bighead encodes a secreted protein that proved to be a potent inhibitor of Wnt signaling in a number of embryological and cultured cell signaling assays. Overexpression of Bighead resulted in large head structures very similar to those of the well-known Wnt antagonists Dkk1 and Frzb-1. Knockdown of Bighead with specific antisense morpholinos resulted in embryos with reduced head structures, due to increased Wnt signaling. Bighead protein bound specifically to the Wnt coreceptor lipoprotein receptor-related protein 6 (Lrp6), leading to its removal from the cell surface. Bighead joins two other Wnt antagonists, Dkk1 and Angptl4, which function as Lrp6 endocytosis regulators. These results suggest that endocytosis plays a crucial role in Wnt signaling.