In amphibians and teleosts, early embryonic axial development is driven by maternally deposited mRNAs and proteins, called dorsal determinants, which migrate to the presumptive dorsal side of the embryo in a microtubule-dependent manner after fertilization. Syntabulin is an adapter protein that binds to kinesin KIF5B and to the transmembrane protein Syntaxin1. In zebrafish, a mutation in Syntabulin causes complete embryo ventralization. It is unknown whether Syntabulin plays an analogous role during early development of other species, a question addressed here in Xenopus laevis. in situ hybridization of syntabulin mRNA was carried out at different stages of Xenopus development. In oocytes, syntabulin transcripts were localized to the vegetal cortex of large oocytes and the mitochondrial cloud of very young oocytes. We extended the zebrafish data by finding that during cleavage Xenopus syntabulin mRNA localized to the germ plasm and was later expressed in primordial germ cells (PGCs). This new finding suggested a role for Syntabulin during germ cell differentiation. The functional role of maternal syntabulin mRNA was investigated by knock-down with phosphorothioate DNA antisense oligos followed by oocyte transfer. The results showed that syntabulin mRNA depletion caused the complete loss of dorso-anterior axis formation in frog embryos. Consistent with the ventralized phenotype, syntabulin-depleted embryos displayed severe reduction of dorsal markers and ubiquitous transcription of the ventral marker sizzled. Syntabulin was required for the maternal Wnt/β-Catenin signal, since ventralization could be completely rescued by injection of β-catenin (or syntabulin) mRNA. The data suggest an evolutionarily conserved role for Syntabulin, a protein that bridges microtubule motors and membrane vesicles, during dorso-ventral axis formation in the vertebrates.

The dorsal half of bisected Xenopus laevis embryos can regenerate a well-proportioned organism on a smaller scale. A new study indicates that the removal of ventral tissue generates a steeper Chordin gradient by reducing Sizzled, a secreted inhibitor of Tolloid chordinases.

Animal embryos have the remarkable property of self-organization. Over 125 years ago, Hans Driesch separated the two blastomeres of sea urchin embryos and obtained twins, in what was the foundation of experimental embryology. Since then, embryonic twinning has been obtained experimentally in many animals. In a recent study, we developed bisection methods that generate identical twins reliably from Xenopus blastula embryos. In the present study, we have investigated the transcriptome of regenerating half-embryos after sagittal and dorsal-ventral (D-V) bisections. Individual embryos were operated at midblastula (stage 8) with an eyelash hair and cultured until early gastrula (stage 10.5) or late gastrula (stage 12) and the transcriptome of both halves were analyzed by RNA-seq. Since many genes are activated by wound healing in Xenopus embryos, we resorted to stringent sequence analyses and identified genes up-regulated in identical twins but not in either dorsal or ventral fragments. At early gastrula, cell division-related transcripts such as histones were elevated, whereas at late gastrula, pluripotency genes (such as sox2) and germ layer determination genes (such as eomesodermin, ripply2 and activin receptor ACVRI) were identified. Among the down-regulated transcripts, sizzled, a regulator of Chordin stability, was prominent. These findings are consistent with a model in which cell division is required to heal damage, while maintaining pluripotency to allow formation of the organizer with a displacement of 90 ⁰ from its original site. The extensive transcriptomic data presented here provides a valuable resource for data mining of gene expression during early vertebrate development.

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.

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 X. laevis complete genome now available provides a powerful tool for unraveling cell-cell signaling pathways during embryonic induction.

Canonical Wnt signaling is emerging as a major regulator of endocytosis. Here, we report that Wnt-induced macropinocytosis is regulated through glycogen synthase kinase 3 (GSK3) and the β-catenin destruction complex. We find that mutation of Axin1, a tumor suppressor and component of the destruction complex, results in the activation of macropinocytosis. Surprisingly, inhibition of GSK3 by lithium chloride (LiCl), CHIR99021, or dominant-negative GSK3 triggers macropinocytosis. GSK3 inhibition causes a rapid increase in acidic endolysosomes that is independent of new protein synthesis. GSK3 inhibition or Axin1 mutation increases lysosomal activity, which can be followed with tracers of active cathepsin D, β-glucosidase, and ovalbumin degradation. Microinjection of LiCl into the blastula cavity of Xenopus embryos causes a striking increase in dextran macropinocytosis. The effects of GSK3 inhibition on protein degradation in endolysosomes are blocked by the macropinocytosis inhibitors EIPA or IPA-3, suggesting that increases in membrane trafficking drive lysosomal activity.

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.

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.

The canonical Wnt pathway serves as a hub connecting diverse cellular processes, including β-catenin signaling, differentiation, growth, protein stability, macropinocytosis, and nutrient acquisition in lysosomes. We have proposed that sequestration of β-catenin destruction complex components in multivesicular bodies (MVBs) is required for sustained canonical Wnt signaling. In this study, we investigated the events that follow activation of the canonical Wnt receptor Lrp6 using an APEX2-mediated proximity labeling approach. The Wnt co-receptor Lrp6 was fused to APEX2 and used to biotinylate targets that are recruited near the receptor during Wnt signaling at different time periods. Lrp6 proximity targets were identified by mass spectrometry, and revealed that many endosomal proteins interacted with Lrp6 within 5 min of Wnt3a treatment. Interestingly, we found that Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, was strongly enriched in the proximity of Lrp6. TFG depletion with siRNA, or knock-out with CRISPR/Cas9, significantly reduced Wnt/β-catenin signaling in cell culture. In vivo, studies in the Xenopus system showed that TFG is required for endogenous Wnt-dependent embryonic patterning. The results suggest that the multivesicular endosomal machinery and the novel player TFG have important roles in Wnt signaling.

Fibroblast growth factor (FGF)/extracellular signal-regulated kinase (ERK) signaling plays a crucial role in anterior-posterior (A-P) axial patterning of vertebrate embryos by promoting posterior development. In our screens for novel developmental regulators in Xenopus embryos, we identified Fam3b as a secreted factor regulated in ectodermal explants. Family with sequence similarity 3 member B (FAM3B)/PANDER (pancreatic-derived factor) is a cytokine involved in glucose metabolism, type 2 diabetes, and cancer in mammals. However, the molecular mechanism of FAM3B action in these processes remains poorly understood, largely because its receptor is still unidentified. Here we uncover an unexpected role of FAM3B acting as a FGF receptor (FGFR) ligand in Xenopus embryos. fam3b messenger RNA (mRNA) is initially expressed maternally and uniformly in the early Xenopus embryo and then in the epidermis at neurula stages. Overexpression of Xenopus fam3b mRNA inhibited cephalic structures and induced ectopic tail-like structures. Recombinant human FAM3B protein was purified readily from transfected tissue culture cells and, when injected into the blastocoele cavity, also caused outgrowth of tail-like structures at the expense of anterior structures, indicating FGF-like activity. Depletion of fam3b by specific antisense morpholino oligonucleotides in Xenopus resulted in macrocephaly in tailbud tadpoles, rescuable by FAM3B protein. Mechanistically, FAM3B protein bound to FGFR and activated the downstream ERK signaling in an FGFR-dependent manner. In Xenopus embryos, FGFR activity was required epistatically downstream of Fam3b to mediate its promotion of posterior cell fates. Our findings define a FAM3B/FGFR/ERK-signaling pathway that is required for axial patterning in Xenopus embryos and may provide molecular insights into FAM3B-associated human diseases.