Description

Previous RNA-sequencing experiments were performed on Xenopus laevis embryos to reveal novel Spemann organizer-specific genes. Analysis on the dorsal and ventral transcriptomes of early frog gastrula revealed an “organizer signature” of genes dorsally expressed and under the control of maternal Wnt signaling (Ding et al., 2017a and b). Among the new genes found in the dorsal side of Xenopus, we identified an additional member of the Dapper antagonist of β-catenin (Dact) family of proteins, called dact4 (Colozza and De Robertis, 2020) as well as several secreted Wnt antagonists, including vlk (Vertebrate lonesome kinase, also known as Pkdcc) (Ding et al., 2017a) angiopoietin-like 4 (Kirsch et al., 2017) and bighead (Ding et al., 2018). The latter represents the only member of a new family of secreted factors conserved in frogs and fishes, but absent in higher vertebrates such as mammals (Ding et al., 2018). Interestingly, protein structure prediction indicated relevant homology with the prodomain region of the latent form of myostatin/growth and differentiation factor 8 (GDF8) (Ding et al., 2018). Xenopus bighead (gene ID 108715768) encodes a Wnt antagonist that binds to the Lrp6 co-receptor and induces its internalization and degradation into lysosomes, downregulating canonical Wnt/β-catenin signaling. Overexpression of bighead mRNA into Xenopus embryos promotes the enlargement of head, eyes and cement gland, accompanied by expanded anterior neural markers, such as rx2a, foxg1 and otx2 (Ding et al., 2018). On the other hand, morpholino-mediated knock-down of bighead has the opposite effect, as shown by the strong reduction in head development and expression of markers such as otx2. Altogether, these evidences suggest that bighead is required for head development in Xenopus embryos via inhibition of Wnt signaling (Ding et al., 2018). Here, I show that secreted Bighead protein purified from the extracellular medium maintains its activity, as assessed in cell culture and Xenopus embryos. To this aim, I used a Streptag/Streptactin-based purification system, which harness the ability of a short tag (8 amino acid long) to bind strongly to modified streptavidin molecules (Maier et al., 1998; Schmidt et al., 1996; Voss and Skerra, 1997). Streptag was introduced at the C-term end of Bighead, where modifications are well tolerated and do not interfere with its signaling activity (Ding et al., 2018). The Streptag was also preceded by a Myc tag, to allow for easier detection of Bighead protein. pCS2 plasmid containing recombinant bighead-Myc-Streptag DNA was transfected into HEK-293T cells. Conditioned medium (CM) containing Bighead protein was collected 48 hours after transfection, and applied to Streptactin columns for affinity purification. Media from empty pCS2 transfected cells was used as a control. Eluted protein was then dialyzed and concentrated in PBS using Millipore concentrators. Western blot analysis confirmed expression and purification of Bighead protein (Fig. 1A). Notably, two major bands were observed between 25 and 37 kDa, suggesting possible post-translational modifications. 200 ng of protein were then applied to the culture medium of HEK-293T cells expressing Lrp6-Flag-APEX2 (Colozza et al., 2020). Compared to control, Lrp6-Flag-APEX2 relocated in intracellular clusters resembling endosomal vesicles, within 3 hours after treatment with purified Bighead protein (Fig. 1B). Interestingly, both Bighead CM and purified protein showed similar effect on Lrp6 subcellular localization, supporting the specificity of Bighead activity. Then, I turned to Xenopus embryos to further characterize the activity of Bighead-Streptag (Fig. 1C). mRNA injections induced strong enlargement of the head and cement gland (Fig. 1D, E), a phenotype that is associated with inhibition of zygotic Wnt signaling (Niehrs, 2001; Ding et al., 2018). Interestingly, injection of the purified protein into the blastocoel (a cavity that collects extracellular fluids and growth factors and plays important role in embryonic patterning) of stage 8 blastula embryos produced similar effects (Fig. 1F-H). This result confirms that purified Bighead protein is active by operating at the extracellular level, as expected for a secreted factor, and promotes head enlargement in Xenopus laevis. This also suggests a possible mechanism for Bighead function in frog embryos, where extracellular Bighead protein (produced and secreted by cells in the Spemann organizer) attenuate Wnt signaling by inducing Lrp6 endocytosis, as proposed in the model in Fig. 1I.

In conclusion, this study provides a straightforward and user-friendly method to purify extracellular soluble factors that can be applied directly to cultured cells and vertebrate embryos to study cell-cell signaling modulation.