Wnt signaling in development and adult tissue homeostasis requires tight regulation to prevent patterning abnormalities and tumor formation. Here, we show that the maternal Wnt antagonist Dkk1 downregulates both the canonical and non-canonical signaling that are required for the correct establishment of the axes of the Xenopus embryo. We find that the target Wnts of Dkk activity are maternal Wnt5a and Wnt11, and that both Wnts are essential for canonical and non-canonical signaling. We determine that Wnt5a and Wnt11 form a previously unrecognized complex. This work suggests a new aspect of Wnt signaling: two Wnts acting in a complex together to regulate embryonic patterning.

Summary Embryo homing and implantation occur within a crypt (implantation chamber) at the antimesometrial (AM) pole along the uterus. The mechanism by which this is achieved is not known. Here we show that villi-like epithelial projections from the main uterine lumen towards the AM pole at regularly spaced intervals to form crypts for embryo implantation were disrupted in mice with uterine loss or gain of function of Wnt5a, or loss of function of both Ror1 and Ror2. This disruption of Wnt5a-ROR signaling resulted in disorderly epithelial projections, crypt formation, embryo spacing, and impaired implantation. These early disturbances under abnormal Wnt5a-ROR signaling were reflected in adverse late pregnancy events, including defective decidualization and placentation, ultimately leading to compromised pregnancy outcome. This study presents deeper insight regarding the formation of organized epithelial projections for crypt formation and embryo implantation for pregnancy success.

Summary An emerging concept in development is that transcriptional poising pre-sets patterns of gene expression in a manner that reflects a cell’s developmental potential. However, it is not known how certain loci are specified in the embryo to establish poised chromatin architecture as the developmental program unfolds. We find that, in the context of transcriptional quiescence prior to the midblastula transition in Xenopus, dorsal specification by the Wnt/β-catenin pathway is temporally uncoupled from the onset of dorsal target gene expression, and that β-catenin establishes poised chromatin architecture at target promoters. β-catenin recruits the arginine methyltransferase Prmt2 to target promoters, thereby establishing asymmetrically dimethylated H3 arginine 8 (R8). Recruitment of Prmt2 to β-catenin target genes is necessary and sufficient to establish the dorsal developmental program, indicating that Prmt2-mediated histone H3R8 methylation plays a critical role downstream of β-catenin in establishing poised chromatin architecture and marking key organizer genes for later expression.

Wnt signaling plays important roles in embryonic development, tissue differentiation, and cancer. In both normal and malignant tissue, Wnt family members are often expressed combinatorially, although the significance of this is not understood. We recently showed that Wnt11 and Wnt5a are both required for the initiation of embryonic axis formation and that the two proteins physically interact with each other. However, little is known about the mechanism or biological significance of Wnt-Wnt protein interaction. Here we show in three assays, with Xenopus oocytes, mouse L cells, and human embryonic stem cells, that secreted Xenopus Wnt11/5a complexes have more canonical Wnt signaling activity than secreted Wnt11 or Wnt5a acting alone. We demonstrate that the sulfation activity of tyrosylprotein sulfotransferase-1 (TPST-1) is required for Xenopus dorsal axis formation and that O-sulfation of specific tyrosine residues is necessary for the interaction of Wnt11 with Wnt5a and for enhanced canonical signaling activity. These findings demonstrate a novel aspect of Wnt biology-Wnt family member interaction that depends on tyrosyl sulfation.

Xvent homeobox genes encode transcription factors that repress organizer genes and are essential for dorsoventral specification during early embryogenesis in Xenopus. In contrast to the Xvent-2 gene subfamily, Xvent-1 subfamily members, including PV.1A, have been proposed as indirect targets of Bone Morphogenetic Protein-4 (BMP-4) signaling. Because PV.1A is a critical downstream mediator of, and tightly regulated by, BMP-4 signaling, we hypothesized that its promoter contains a direct BMP-4 response element to effect this transcriptional regulation. We demonstrate that direct regulation by BMP-4 is necessary for transcription of PV.1A: its proximal promoter contains cis-acting binding elements for Smads and Oaz crucial to induction in response to BMP-4 signaling. In addition to these direct cis-acting BMP-4 responsive elements, an indirect Xvent-2 response element and several repressive elements exist in the PV.1A promoter to regulate its transcription. In summary, PV.1A undergoes combinatorial regulation during early Xenopus development as both the direct target of BMP-4 signaling and as the direct and indirect target of positive and negative regulatory factors.

The revolution in CRISPR-mediated genome editing has enabled the mutation and insertion of virtually any DNA sequence, particularly in cell culture where selection can be used to recover relatively rare homologous recombination events. The efficient use of this technology in animal models still presents a number of challenges, including the time to establish mutant lines, mosaic gene editing in founder animals, and low homologous recombination rates. Here we report a method for CRISPR-mediated genome editing in Xenopus oocytes with homology-directed repair (HDR) that provides efficient non-mosaic targeted insertion of small DNA fragments (40-50 nucleotides) in 4.4-25.7% of F0 tadpoles, with germline transmission. For both CRISPR/Cas9-mediated HDR gene editing and indel mutation, the gene-edited F0 embryos are uniformly heterozygous, consistent with a mutation in only the maternal genome. In addition to efficient tagging of proteins in vivo, this HDR methodology will allow researchers to create patient-specific mutations for human disease modeling in Xenopus.

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The trachea and esophagus arise from the separation of a common foregut tube during early fetal development. Mutations in key signaling pathways such as Hedgehog (HH)/Gli can disrupt tracheoesophageal (TE) morphogenesis and cause life-threatening birth defects (TEDs); however, the underlying cellular mechanisms are unknown. Here, we use mouse and Xenopus to define the HH/Gli-dependent processes orchestrating TE morphogenesis. We show that downstream of Gli the Foxf1+ splanchnic mesenchyme promotes medial constriction of the foregut at the boundary between the presumptive Sox2+ esophageal and Nkx2-1+ tracheal epithelium. We identify a unique boundary epithelium co-expressing Sox2 and Nkx2-1 that fuses to form a transient septum. Septum formation and resolution into distinct trachea and esophagus requires endosome-mediated epithelial remodeling involving the small GTPase Rab11 and localized extracellular matrix degradation. These are disrupted in Gli-deficient embryos. This work provides a new mechanistic framework for TE morphogenesis and informs the cellular basis of human TEDs.