Highlights d GSK3 and Axin1 normally repress macropinocytosis in basal cellular conditions d GSK3 inhibition or Axin1 mutation triggers macropinocytosis and metabolite changes d Macropinocytosis induces acidification and catabolic activity of lysosomes d Lysosomal activation by Wnt signaling is independent of new protein synthesis

The earliest event in Xenopus development is the dorsal accumulation of nuclear β-catenin under the influence of cytoplasmic determinants displaced by fertilization. In this study, a genome-wide approach was used to examine transcription of the 43,673 genes annotated in the Xenopus laevis genome under a variety of conditions that inhibit or promote formation of the Spemann organizer signaling center. Loss of function of β-catenin with antisense morpholinos reproducibly reduced the expression of 247 mRNAs at gastrula stage. Interestingly, only 123 β-catenin targets were enriched on the dorsal side and defined an early dorsal β-catenin gene signature. These genes included several previously unrecognized Spemann organizer components. Surprisingly, only 3 of these 123 genes overlapped with the late Wnt signature recently defined by two other groups using inhibition by Dkk1 mRNA or Wnt8 morpholinos, which indicates that the effects of β-catenin/ Wnt signaling in early development are exquisitely regulated by stage-dependent mechanisms. We analyzed transcriptome responses to a number of treatments in a total of 46 RNA-seq libraries. These treatments included, in addition to β-catenin depletion, regenerating dorsal and ventral half-embryos, lithium chloride treatment, and the overexpression of Wnt8, Siamois, and Cerberus mRNAs. Only some of the early dorsal β-catenin signature genes were activated at blastula whereas others required the induction of endomesoderm, as indicated by their inhibition by Cerberus overexpression. These comprehensive data provide a rich resource for analyzing how the dorsal and ventral regions of the embryo communicate with each other in a self-organizing vertebrate model embryo.

RNA sequencing has allowed high-throughput screening of differential gene expression in many tissues and organisms. Xenopus laevis is a classical embryological and cell-free extract model system, but its genomic sequence had been lacking due to difficulties arising from allotetraploidy. There is currently much excitement surrounding the release of the completed X. laevis genome (version 9.1) by the Joint Genome Institute (JGI), which provides a platform for genome-wide studies. Here we present a deep RNA-seq dataset of transcripts expressed in dorsal and ventral lips of the early Xenopus gastrula embryo using the new genomic information, which was further annotated by blast searches against the human proteome. Overall, our findings confirm previous results from differential screenings using other methods that uncovered classical dorsal genes such as Chordin, Noggin and Cerberus, as well as ventral genes such as Sizzled, Ventx, Wnt8 and Bambi. Complete transcriptome-wide tables of mRNAs suitable for data mining are presented, which include many novel dorsal- and ventral-specific genes. RNA-seq was very quantitative and reproducible, and allowed us to define dorsal and ventral signatures useful for gene set expression analyses (GSEA). As an example of a new gene, we present here data on an organizer-specific secreted protein tyrosine kinase known as Pkdcc (protein kinase domain containing, cytoplasmic) or Vlk (vertebrate lonesome kinase). Overexpression experiments indicate that Pkdcc can act as a negative regulator of Wnt/ β-catenin signaling independently of its kinase activity. We conclude that RNA-Seq in combination with the Xenopus laevis complete genome now available provides a powerful tool for unravelling cell-cell signaling pathways during embryonic induction.

Angiopoietin-like 4 (ANGPTL4) is a secreted signaling protein that is implicated in cardiovascular disease, metabolic disorder, and cancer. Outside of its role in lipid metabolism, ANGPTL4 signaling remains poorly understood. Here, we identify ANGPTL4 as a Wnt signaling antagonist that binds to syndecans and forms a ternary complex with the Wnt co-receptor Lipoprotein receptor-related protein 6 (LRP6). This protein complex is internalized via clathrin-mediated endocytosis and degraded in lysosomes, leading to attenuation of Wnt/β-catenin signaling. Angptl4 is expressed in the Spemann organizer of Xenopus embryos and acts as a Wnt antagonist to promote notochord formation and prevent muscle differentiation. This unexpected function of ANGPTL4 invites re-interpretation of its diverse physiological effects in light of Wnt signaling and may open therapeutic avenues for human disease.

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A striking aspect of tissue regeneration is its uneven distribution among different animal classes, both in terms of modalities and efficiency. The retina does not escape the rule, exhibiting extraordinary self-repair properties in anamniote species but extremely limited ones in mammals. Among cellular sources prone to contribute to retinal regeneration are Müller glial cells, which in teleosts have been known for a decade to re-acquire a stem/progenitor state and regenerate retinal neurons following injury. As their regenerative potential was hitherto unexplored in amphibians, we tackled this issue using two Xenopus retinal injury paradigms we implemented: a mechanical needle poke injury and a transgenic model allowing for conditional photoreceptor cell ablation. These models revealed that Müller cells are indeed able to proliferate and replace lost cells following damage/degeneration in the retina. Interestingly, the extent of cell cycle re-entry appears dependent on the age of the animal, with a refractory period in early tadpole stages. Our findings pave the way for future studies aimed at identifying the molecular cues that either sustain or constrain the recruitment of Müller glia, an issue of utmost importance to set up therapeutic strategies for eye regenerative medicine.

SignificanceThe 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.

Wnt signaling is an evolutionarily conserved pathway involved in embryonic development and cell differentiation (Nusse & Clevers, 2017).In addition, it regulates many other processes, including cell growth and mitosis, adult stem cell homeostasis, and regeneration (Acebron & Niehrs, 2016;Nusse & Clevers, 2017;Reddien, 2018). Aberrant activation of the Wnt pathway lies at the basis of different types of malignancies, including colorectal, breast, and hepatic cancers.Historically, the first Wnt gene was identified in Drosophila melanogaster, following a screen for recessive mutant genes that affect segment patterning of fruit fly embryos. Because the mutant flies did not develop wings, the gene was named wingless (wg) (Sharma, 1973;Sharma & Chopra, 1976). Years later, the proto-oncogene integration site-1 (int-1) was discovered while studying mouse models of mammary carcinomas induced by DNA integrations of the mouse mammary tumor virus (MMTV) (Nusse & Varmus, 1982). Since Int-1 was found to be the mammalian orthologue of Wg, it was then renamed Wnt1 (Wingless + Int1) (Nusse et al., 1991).Wnts are secreted glycoproteins (Brown et al., 1987;Papkoff et al., 1987) that can activate divergent pathways, classically categorized as: (a) the canonical or β-catenin dependent pathway; and (b) the noncanonical branch, comprising the planar cell polarity and Ca 2+ pathways. Although some Wnts preferentially activate one or the other pathway, their specificity is less strict than previously thought and likely depends on the cell-specific context and the repertoire of receptors and components expressed, rather than on intrinsic properties. Indeed, while the serpentine Frizzled (Fzd) receptors are a shared component of all Wnt pathways, coreceptors such as the low-density lipoprotein receptor-related protein 5 and 6 (Lrp5/6;