How to Generate Non-Mosaic CRISPR/Cas9 Mediated Knock-In and Mutations in F0 Xenopus Through the Host-Transfer Technique.

We have taken advantage of the well-established oocyte host transfer technique to optimize a method for CRISPR editing of Xenopus that provides an efficient non-mosaic targeted insertion of small DNA fragment through homology-directed repair mechanism.

High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus.

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.

Nodal signalling in Xenopus: the role of Xnr5 in left/right asymmetry and heart development.

Nodal class TGF-ß signalling molecules play essential roles in establishing the vertebrate body plan. In all vertebrates, nodal family members have specific waves of expression required for tissue specification and axis formation. In Xenopus laevis, six nodal genes are expressed before gastrulation, raising the question of whether they have specific roles or act redundantly with each other. Here, we examine the role of Xnr5. We find it acts at the late blastula stage as a mesoderm inducer and repressor of ectodermal gene expression, a role it shares with Vg1. However, unlike Vg1, Xnr5 depletion reduces the expression of the nodal family member xnr1 at the gastrula stage. It is also required for left/right laterality by controlling the expression of the laterality genes xnr1, antivin (lefty) and pitx2 at the tailbud stage. In Xnr5-depleted embryos, the heart field is established normally, but symmetrical reduction in Xnr5 levels causes a severely stunted midline heart, first evidenced by a reduction in cardiac troponin mRNA levels, while left-sided reduction leads to randomization of the left/right axis. This work identifies Xnr5 as the earliest step in the signalling pathway establishing normal heart laterality in Xenopus.

The Eyes Absent Proteins in Developmental and Pathological Angiogenesis.

Management of neoangiogenesis remains a high-value therapeutic goal. A recently uncovered association between the DNA damage repair pathway and pathological angiogenesis could open previously unexplored possibilities for intervention. An attractive and novel target is the Eyes absent (EYA) tyrosine phosphatase, which plays a critical role in the repair versus apoptosis decision after DNA damage. This study examines the role of EYA in the postnatal development of the retinal vasculature and under conditions of ischemia-reperfusion encountered in proliferative retinopathies. We find that the ability of the EYA proteins to promote endothelial cell (EC) migration contributes to a delay in postnatal development of the retinal vasculature when Eya3 is deleted specifically in ECs. By using genetic and chemical biology tools, we show that EYA contributes to pathological angiogenesis in a model of oxygen-induced retinopathy. Both in vivo and in vitro, loss of EYA tyrosine phosphatase activity leads to defective assembly of γ-H2AX foci and thus to DNA damage repair in ECs under oxidative stress. These data reveal the potential utility of EYA tyrosine phosphatase inhibitors as therapeutic agents in inhibiting pathological neovascularization with a range of clinical applications.

Structure-activity relationships of benzbromarone metabolites and derivatives as EYA inhibitory anti-angiogenic agents.

The tyrosine phosphatase activity of the phosphatase-transactivator protein Eyes Absent (EYA) is angiogenic through its roles in endothelial cell migration and tube formation. Benzbromarone, a known anti-gout agent, was previously identified as an inhibitor of EYA with anti-angiogenic properties. Here we show that the major metabolite of BBR, 6-hydroxy benzbromarone, is a significantly more potent inhibitor of cell migration, tubulogenesis and angiogenic sprouting. In contrast, other postulated metabolites of BBR such as 5-hydroxy benzbromaorne and 1'-hydroxy benzbromarone are less potent inhibitors of EYA tyrosine phosphatase activity as well as being less effective in cellular assays for endothelial cell migration and angiogenesis. Longer substituents at the 2 position of the benzofuran ring promoted EYA3 binding and inhibition, but were less effective in cellular assays, likely reflecting non-specific protein binding and a resulting reduction in free, bio-available inhibitor. The observed potency of 6-hydroxy benzbromarone is relevant in the context of the potential re-purposing of benzbromarone and its derivatives as anti-angiogenic agents. 6-hydroxy benzbromarone represents a metabolite with a longer half-life and greater pharmacological potency than the parent compound, suggesting that biotransformation of benzbromarone could contribute to its therapeutic activity.

The Eyes Absent proteins in development and disease.

The Eyes Absent (EYA) proteins, first described in the context of fly eye development, are now implicated in processes as disparate as organ development, innate immunity, DNA damage repair, photoperiodism, angiogenesis, and cancer metastasis. These functions are associated with an unusual combination of biochemical activities: tyrosine phosphatase and threonine phosphatase activities in separate domains, and transactivation potential when associated with a DNA-binding partner. EYA mutations are linked to multiorgan developmental disorders, as well as to adult diseases ranging from dilated cardiomyopathy to late-onset sensorineural hearing loss. With the growing understanding of EYA biochemical and cellular activity, biological function, and association with disease, comes the possibility that the EYA proteins are amenable to the design of targeted therapeutics. The availability of structural information, direct links to disease states, available animal models, and the fact that they utilize unconventional reaction mechanisms that could allow specificity, suggest that EYAs are well-positioned for drug discovery efforts. This review provides a summary of EYA structure, activity, and function, as they relate to development and disease, with particular emphasis on recent findings.

The EYA tyrosine phosphatase activity is pro-angiogenic and is inhibited by benzbromarone.

Eyes Absents (EYA) are multifunctional proteins best known for their role in organogenesis. There is accumulating evidence that overexpression of EYAs in breast and ovarian cancers, and in malignant peripheral nerve sheath tumors, correlates with tumor growth and increased metastasis. The EYA protein is both a transcriptional activator and a tyrosine phosphatase, and the tyrosine phosphatase activity promotes single cell motility of mammary epithelial cells. Since EYAs are expressed in vascular endothelial cells and cell motility is a critical feature of angiogenesis we investigated the role of EYAs in this process. Using RNA interference techniques we show that EYA3 depletion in human umbilical vein endothelial cells inhibits transwell migration as well as Matrigel-induced tube formation. To specifically query the role of the EYA tyrosine phosphatase activity we employed a chemical biology approach. Through an experimental screen the uricosuric agents Benzbromarone and Benzarone were found to be potent EYA inhibitors, and Benzarone in particular exhibited selectivity towards EYA versus a representative classical protein tyrosine phosphatase, PTP1B. These compounds inhibit the motility of mammary epithelial cells over-expressing EYA2 as well as the motility of endothelial cells. Furthermore, they attenuate tubulogenesis in matrigel and sprouting angiogenesis in the ex vivo aortic ring assay in a dose-dependent fashion. The anti-angiogenic effect of the inhibitors was also demonstrated in vivo, as treatment of zebrafish embryos led to significant and dose-dependent defects in the developing vasculature. Taken together our results demonstrate that the EYA tyrosine phosphatase activity is pro-angiogenic and that Benzbromarone and Benzarone are attractive candidates for repurposing as drugs for the treatment of cancer metastasis, tumor angiogenesis, and vasculopathies.

The roles of maternal Vangl2 and aPKC in Xenopus oocyte and embryo patterning.

The Xenopus oocyte contains components of both the planar cell polarity and apical-basal polarity pathways, but their roles are not known. Here, we examine the distribution, interactions and functions of the maternal planar cell polarity core protein Vangl2 and the apical-basal complex component aPKC. We show that Vangl2 is distributed in animally enriched islands in the subcortical cytoplasm in full-grown oocytes, where it interacts with a post-Golgi v-SNARE protein, VAMP1, and acetylated microtubules. We find that Vangl2 is required for the stability of VAMP1 as well as for the maintenance of the stable microtubule architecture of the oocyte. We show that Vangl2 interacts with atypical PKC, and that both the acetylated microtubule cytoskeleton and the Vangl2-VAMP1 distribution are dependent on the presence of aPKC. We also demonstrate that aPKC and Vangl2 are required for the cell membrane asymmetry that is established during oocyte maturation, and for the asymmetrical distribution of maternal transcripts for the germ layer and dorsal/ventral determinants VegT and Wnt11. This study demonstrates the interaction and interdependence of Vangl2, VAMP1, aPKC and the stable microtubule cytoskeleton in the oocyte, shows that maternal Vangl2 and aPKC are required for specific oocyte asymmetries and vertebrate embryonic patterning, and points to the usefulness of the oocyte as a model to study the polarity problem.

The functions of maternal Dishevelled 2 and 3 in the early Xenopus embryo.

Of the three Dishevelled (Dvl) genes, only Dvl2 and Dvl3 are maternally encoded in the frog, Xenopus laevis. We show here by loss of function analysis that single depletion of either Dvl2 or Dvl3 from the oocyte causes the same embryonic phenotype. We find that the effects of loss of function of Dvl2 and 3 together are additive, and that the proteins physically interact, suggesting that both are required in the same complex. We show that maternal Dvl2 and 3 are required for convergence extension movements downstream of the dorsally localized signaling pathway activated by Xnr3, but not downstream of the pathway activated by activin. Also, depletion of maternal Dvl2 and 3 mRNAs causes the up-regulation of a subset of zygotic ectodermal genes, including Foxi1e, with surprisingly no significant effect on the canonical Wnt direct target genes Siamois and Xnr3. We suggest that the likely reason for continued expression of the Wnt target genes in Dvl2/3-depleted embryos is that maternal Dvl mRNA depletion is insufficient to deplete stored punctae of Dvl protein in the oocyte cortex, which may transduce dorsal signaling after fertilization.

beta-Catenin primes organizer gene expression by recruiting a histone H3 arginine 8 methyltransferase, Prmt2.

An emerging concept in development is that transcriptional poising presets 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/beta-catenin pathway is temporally uncoupled from the onset of dorsal target gene expression, and that beta-catenin establishes poised chromatin architecture at target promoters. beta-catenin recruits the arginine methyltransferase Prmt2 to target promoters, thereby establishing asymmetrically dimethylated H3 arginine 8 (R8). Recruitment of Prmt2 to beta-catenin target genes is necessary and sufficient to establish the dorsal developmental program, indicating that Prmt2-mediated histone H3(R8) methylation plays a critical role downstream of beta-catenin in establishing poised chromatin architecture and marking key organizer genes for later expression.

A co-dependent requirement of xBcl9 and Pygopus for embryonic body axis development in Xenopus.

The Wnt/beta-catenin transcriptional activation complex requires the adapter protein Pygopus (Pygo), which links the basal transcription machinery to beta-catenin, by its association with legless (Lgs)/ B-cell lymphoma-9 (Bcl9). Pygo was shown to be required for development in vertebrates, but the role of Lgs/Bcl9 is unknown. We identified an amphibian orthologue of Lgs/Bcl9, XBcl9, which interacted biochemically with Xbeta-catenin and XPygo2. The body axis promoting ability of Xbeta-catenin was diminished when residues required for its interaction with XBcl9 were mutated. In blastula embryos, XBcl9 was transiently preferentially expressed in nuclei of dorsoanterior cells and ectopically expressed XBcl9 required XPygo2 to localize to nuclei. Furthermore, while neither XBcl9 nor XPygo2 alone affected development when ectopically expressed, both were required to induce supernumerary axis and dorsal gene activation. Like XPygo2, depletion of maternal XBcl9 alone caused dorsal defects. These results indicated an essential role of the Pygo-Bcl9 duet in vertebrate body axis formation.

Wnt11/5a complex formation caused by tyrosine sulfation increases canonical signaling activity.

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.

Wnt5a and Wnt11 interact in a maternal Dkk1-regulated fashion to activate both canonical and non-canonical signaling in Xenopus axis formation.

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.

Manipulation of hedgehog signaling in Xenopus by means of embryo microinjection and application of chemical inhibitors.

Xenopus embryos provide a powerful model system to investigate the complex molecular mechanisms, which are controlled by or control the activity of the Hedgehog (Hh) signaling pathway. The use of synthetic mRNA or antisense oligonucleotide (morpholino) microinjection into blastomeres of early embryos or by simply treating the embryos with small organic inhibitors, has already led to an idea of the network in which the Hh pathway is embedded. More needs to be done in order to achieve a detailed understanding of how the different players of the Hh signaling pathway are integrated to control different genetic programs, such as axis formation in early embryos or cell differentiation during retinogenesis.

Cholesterol homeostasis in development: the role of Xenopus 7-dehydrocholesterol reductase (Xdhcr7) in neural development.

7-dehydrocholesterol reductase (7-Dhcr) catalyses the final step in the pathway of cholesterol biosynthesis. Human patients with inborn errors of 7-Dhcr (Smith-Lemli-Opitz-Syndrome) have elevated serum levels of 7-dehydrocholesterol but low levels of cholesterol, which in phenotypical terms can result in growth retardation, craniofacial abnormalities including cleft palate, and reduced metal abilities. This study reports the isolation and molecular characterisation of 7-dehydrocholesterol reductase (Xdhcr7) from Xenopus laevis. During early embryonic development, the expression of Xdhcr7 is first of all spatially restricted to the Spemann's organizer and later to the notochord. In both tissues, Xdhcr7 is coexpressed with Sonic hedgehog (Shh), which itself is cholesterol-modified during autoproteolytic cleavage. Data from Xdhcr7 overexpression and knockdown experiments reveals that a tight control of cholesterol synthesis is particularly important for proper development of the central and peripheral nervous system.