Embryotoxicity of statins and other prescribed drugs with reported off-target effects on cholesterol biosynthesis.

Cholesterol plays pivotal cellular functions ranging from maintaining membrane fluidity to regulating cell-cell signaling. High cholesterol causes cardiovascular diseases, low cholesterol is linked to neuropsychiatric disorders, and inborn errors of cholesterol synthesis cause multisystem malformation syndromes. Statins lower cholesterol levels by inhibiting the first, rate-limiting reaction of the cholesterol biosynthesis pathway catalyzed by hydroxymethyl-glutaryl-Coenzyme A reductase (HMGCR).

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 .

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 oocytes with homology-directed repair (HDR) that provides efficient non-mosaic targeted insertion of small DNA fragments (40-50 nucleotides) in 4.

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

Nodal class TGF-β 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.