Antiangiogenic and Neurogenic Activities of Sleeping Beauty-Mediated PEDF-Transfected RPE Cells In Vitro and In Vivo.

Pigment epithelium-derived factor (PEDF) is a potent multifunctional protein that inhibits angiogenesis and has neurogenic and neuroprotective properties. Since the wet form of age-related macular degeneration is characterized by choroidal neovascularization (CNV), PEDF would be an ideal candidate to inhibit CNV and support retinal pigment epithelial (RPE) cells. However, its short half-life has precluded its clinical use.

LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling.

R-spondins are secreted Wnt signalling agonists, which regulate embryonic patterning and stem cell proliferation, but whose mechanism of action is poorly understood. Here we show that R-spondins bind to the orphan G-protein-coupled receptors LGR4 and LGR5 by their Furin domains. Gain- and loss-of-function experiments in mammalian cells and Xenopus embryos indicate that LGR4 and LGR5 promote R-spondin-mediated Wnt/β-catenin and Wnt/PCP signalling.

Endogenic regulation of proliferation and zinc transporters by pigment epithelial cells nonvirally transfected with PEDF.

Purpose: Genetic modification of cells before transplantation may allow the delivery of neuroprotective and other functional molecules to patients with neurodegenerative diseases. To avoid complications associated with virally transfected cells, we have explored the use of nonviral methods to insert genetic material into RPE cells.

Methods: After transfection with plasmids encoding different pigment epithelium-derived factor (PEDF) fusion proteins, transfected cells were established and passaged up to 100 times.

The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development.

The vertebrate embryonic vasculature develops from angioblasts, which are specified from mesodermal precursors and develop in close association with blood cells. The signals that regulate embryonic vasculogenesis and angiogenesis are incompletely understood. Here, we show that R-spondin 3 (Rspo3), a member of a novel family of secreted proteins in vertebrates that activate Wnt/beta-catenin signaling, plays a key role in these processes.

Dickkopf-1 regulates gastrulation movements by coordinated modulation of Wnt/beta catenin and Wnt/PCP activities, through interaction with the Dally-like homolog Knypek.

Dickkopf-1 (Dkk1) is a secreted protein that negatively modulates the Wnt/beta catenin pathway. Lack of Dkk1 function affects head formation in frog and mice, supporting the idea that Dkk1 acts as a "head inducer" during gastrulation. We show here that lack of Dkk1 function accelerates internalization and rostral progression of the mesendoderm and that gain of function slows down both internalization and convergence extension, indicating a novel role for Dkk1 in modulating these movements.

R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis.

We have carried out a small pool expression screen for modulators of the Wnt/beta-catenin pathway and identified Xenopus R-spondin2 (Rspo2) as a secreted activator of this cascade. Rspo2 is coexpressed with and positively regulated by Wnt signals and synergizes with Wnts to activate beta-catenin. Analyses of functional interaction with components of the Wnt/beta-catenin pathway suggest that Rspo2 functions extracellularly at the level of receptor ligand interaction.

Characterization of cis-regulatory elements of the homeobox gene Xanf-1.

Investigation of molecular mechanisms underlying early patterning of the nervous system is an important task of modern developmental biology. Previously, we identified a novel homeobox gene, Anf, that is expressed in the most anterior zone at the beginning of neuroectoderm specification. The expression pattern of Anf corresponds to primordia of the telencephalon and the rostral part of the diencephalon.

Dickkopf1 and the Spemann-Mangold head organizer.

Work in amphibians indicates that inhibition of Wnt and BMP signals is essential for head development and that head induction by the Spemann-Mangold organizer may be mediated by secreted Wnt antagonists. Wnts are potent posteriorizing factors and antagonize the Spemann-Mangold organizer. Dickkopf1 (dkk1) encodes a secreted effector expressed in head organizing centers of Xenopus, mouse and zebrafish.

The role of Xenopus dickkopf1 in prechordal plate specification and neural patterning.

Dickkopf1 (dkk1) encodes a secreted WNT inhibitor expressed in Spemann's organizer, which has been implicated in head induction in Xenopus. Here we have analyzed the role of dkk1 in endomesoderm specification and neural patterning by gain- and loss-of-function approaches. We find that dkk1, unlike other WNT inhibitors, is able to induce functional prechordal plate, which explains its ability to induce secondary heads with bilateral eyes.

The homeobox gene, Xanf-1, can control both neural differentiation and patterning in the presumptive anterior neurectoderm of the Xenopus laevis embryo.

From the onset of neurectoderm differentiation, homeobox genes of the Anf class are expressed within a region corresponding to the presumptive telencephalic and rostral diencephalic primordia. Here we investigate functions of the Xenopus member of Anf, Xanf-1, in the differentiation of the anterior neurectoderm. We demonstrate that ectopic Xanf-1 can expand the neural plate at expense of adjacent non-neural ectoderm.

A novel marker of early epidermal differentiation: cDNA subtractive cloning starting on a single explant of Xenopus laevis gastrula epidermis.

To understand the molecular mechanism underlying in the earliest steps of the embryonic ectoderm subdivision into epidermis and neuroectoderm, it would be important to isolate differentially expressed genes in presumptive neuroectoderm and epidermis at the gastrula stage, the period of the divergence of the two adjacent ectodermal compartments. Meanwhile, the most direct approach for such a task, i.e.

Anf: a novel class of vertebrate homeobox genes expressed at the anterior end of the main embryonic axis.

Five novel genes homologous to the homeobox-containing genes Xanf-1 and Xanf-2 of Xenopus and Hesx-1/Rpx of mouse have been identified as a result of a PCR survey of cDNA in sturgeon, zebrafish, newt, chicken and human. Comparative analysis of the homeodomain primary structure of these genes revealed that they belong to a novel class of homeobox genes, which we name Anf. All genes of this class investigated so far have similar patterns of expression during early embryogenesis, characterized by maximal transcript levels being present at the anterior extremity of the main embryonic body axis.

The homeobox-containing gene XANF-1 may control development of the Spemann organizer.

At the beginning of gastrulation the homeobox-containing gene, XANF-1, is expressed at a low level throughout the animal hemisphere of Xenopus laevis embryos, with a local maximum of expression in the region of the dorsal blastopore lip. By the end of gastrulation expression ceases everywhere except in the most anterior part of the neurectoderm. We have investigated the functions of this gene by microinjecting XANF-1 mRNA in the blastomeres of the 32-cell stage embryo and have observed the following effects.

A novel homeobox gene expressed in the anterior neural plate of the Xenopus embryo.

To obtain gene sequences controlling the early steps of amphibian neurogenesis, we have performed differential screening of a subtractive cDNA library prepared by a novel PCR-based method from a single presumptive neural plate of a Xenopus laevis late-gastrula embryo. As a result we have isolated a fragment of a novel homeobox gene (named XANF-1, for Xenopus anterior neural folds). This gene is expressed predominantly in the anterior part of the developing nervous system.