Purified Bighead protein efficiently promotes head development in the South African clawed frog, Xenopus laevis

Abstract

Vertebrate embryonic development is regulated by a few families of extracellular signaling molecules. Xenopus laevis embryos offer an excellent system to study the cell-cell communication signals that govern embryonic patterning. In the frog embryos, Wnt/β-catenin plays a pivotal role in regulating embryonic axis development, and modulation of the Wnt pathway is required for proper antero-posterior patterning. Recently, a novel secreted, organizer-specific Wnt inhibitor, Bighead, was identified that acts by downregulating Lrp6 plasma membrane levels. Here, I describe a method to purify biologically active Bighead protein and confirm that Bighead promotes Xenopus head development.

Figure 1. Purified Bighead Strep-Tag induces Lrp6 relocalization and promotes expansion of anterior tissues in Xenopus laevis

A) Western blot confirming Bighead-Myc Streptag purification from extracellular medium. HEK-293T cells were transfected with Bighead-Myc Streptag plasmid. Conditioned medium containing Bighead Streptag was collected from transfected cells and affinity purified by using streptactin columns. Purification of secreted Bighead protein was assessed using an anti-Myc antibody. Bighead was detected only in transfected cells (lanes 2 and 4) and was absent in control cells (lanes 1 and 3). Note that two major bands were observed, between 25 and 37 kDa, both in whole cell lysate and after purification. These may reflect different post-translational modifications that affect protein molecular weight and, hence, electrophoretic migration.Tubulin, present just in the cell lysate, was used as a loading control. B) When applied to the culture medium of HEK-293T cells expressing a chimeric Lrp6-Flag Apex2 construct, purified Bighead-Myc Streptag induces Lrp6 relocalization in intracellular puncta, reminiscent of endosomal vesicles, as compared to control cells treated with PBS only. A similar effect is obtained when cells are incubated with conditioned medium obtained from Bighead expressing cells (BH CM). Note how Lrp6 changes its localization from the plasma membrane (cells treated with control media; see arrowheads) to intracellular vesicles that tend to concentrate in the nuclear bay (arrowheads). Scale bars represent 10 μm. C) Cartoon depicting embryo injections with bighead-myc streptag mRNA (left) or purified protein (right). D-G) Frontal view of embryos injected as shown in panel C, and collected at tailbud stage (st. 22). 200 ng of bighead-myc streptag mRNA were injected in each blastomere of Xenopus embryos at the 4-cell stage. Injected embryos show clear expansion of anterior structures, such as the cement gland (E, black arrowheads pointing at cement gland borders), as compared to uninjected WT embryos (D). Embryos injected with purified Bighead protein inside the blastocoel cavity at stage 8 (G), displayed head enlargement compared to PBS-injected controls (F), akin to mRNA injections. Scale bar in D represents 500 μm. All embryos were photographed at the same magnification. H) Quantifications of the experiment shown in D-G. Plotted are the percentages of embryos showing anteriorization phenotype (evaluated by the enlargement of head and cement gland). The total number (n) of embryos used for this experiment is shown in each column. I) Cartoon showing the proposed model for Bighead activity during patterning of the Xenopus gastrula. In vivo, Bighead is secreted by the Spemann organizer, where it represses Wnt signaling by binding to Lrp6 receptor and inducing its internalization.