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Using Confocal Analysis of <em>Xenopus laevis</em> to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Newcastle University

Cyclin E supports pre-replication complex (pre-RC) assembly, while cyclin A-associated kinase activates DNA synthesis. We show that cyclin E, but not A, is mounted upon the nuclear matrix in sub-nuclear foci in differentiated vertebrate cells, but not in undifferentiated cells or cancer cells. In murine embryonic stem cells, Xenopus embryos and human urothelial cells, cyclin E is recruited to the nuclear matrix as cells differentiate and this can be manipulated in vitro. This suggests that pre-RC assembly becomes spatially restricted as template usage is defined. Furthermore, failure to become restricted may contribute to the plasticity of cancer cells.

Cyclin E is recruited to the nuclear matrix during differentiation, but is not recruited in cancer cells.

We have previously shown that the Gsx family homeobox gene Gsh2 is part of the regulatory network specifying dorsoventral pattern of primary neurons in the developing amphibian embryo. Here, we investigate the role of Gsx transcription factors in regulating the transcription of Iroquois family homeobox genes in the amphibian neural plate. Iroquois genes are key regulators of neural patterning and their expression is coincident with that of the Gsx genes during open neural plate stages. We show that Gsx proteins repress Iroquois expression in the embryo and conversely, inhibition of Gsx activity with either antisense morpholino oligos or an anti-morphic Gsx protein up-regulates Iroquois expression. These data indicate that Gsx factors act as negative regulators of Iroquois gene expression in the amphibian neural plate and support a model in which the Gsx proteins promote neuronal differentiation by repressing the expression of known inhibitors of neuronal differentiation such as Iro3.

Gsx transcription factors repress Iroquois gene expression.

Lin28 family proteins share a unique structure, with both zinc knuckle and cold shock RNA-binding domains, and were originally identified as regulators of developmental timing in Caenorhabditis elegans. They have since been implicated as regulators of pluripotency in mammalian stem cells in culture. Using Xenopus tropicalis, we have undertaken the first analysis of the effects on the early development of a vertebrate embryo resulting from global inhibition of the Lin28 family. The Xenopus genome contains two Lin28-related genes, lin28a and lin28b. lin28a is expressed zygotically, whereas lin28b is expressed both zygotically and maternally. Both lin28a and lin28b are expressed in pluripotent cells of the Xenopus embryo and are enriched in cells that respond to mesoderm-inducing signals. The development of axial and paraxial mesoderm is severely abnormal in lin28 knockdown (morphant) embryos. In culture, the ability of pluripotent cells from the embryo to respond to the FGF and activin/nodal-like mesoderm-inducing pathways is compromised following inhibition of lin28 function. Furthermore, there are complex effects on the temporal regulation of, and the responses to, mesoderm-inducing signals in lin28 morphant embryos. We provide evidence that Xenopus lin28 proteins play a key role in choreographing the responses of pluripotent cells in the early embryo to the signals that regulate germ layer specification, and that this early function is probably independent of the recognised role of Lin28 proteins in negatively regulating let-7 miRNA biogenesis.

Lin28 proteins are required for germ layer specification in Xenopus.

Genetic studies have established that heparan sulphate proteoglycans (HSPGs) are required for signalling by key developmental regulators, including Hedgehog, Wnt/Wg, FGF, and BMP/Dpp. Post-synthetic remodelling of heparan sulphate (HS) by Sulf1 has been shown to modulate these same signalling pathways. Sulf1 codes for an N-acetylglucosamine 6-O-endosulfatase, an enzyme that specifically removes the 6-O sulphate group from glucosamine in highly sulfated regions of HS chains. One striking aspect of Sulf1 expression in all vertebrates is its co-localisation with that of Sonic hedgehog in the floor plate of the neural tube. We show here that Sulf1 is required for normal specification of neural progenitors in the ventral neural tube, a process known to require a gradient of Shh activity. We use single-cell injection of mRNA coding for GFP-tagged Shh in early Xenopus embryos and find that Sulf1 restricts ligand diffusion. Moreover, we find that the endogenous distribution of Shh protein in Sulf1 knockdown embryos is altered, where a less steep ventral to dorsal gradient forms in the absence of Sulf1, resulting in more a diffuse distribution of Shh. These data point to an important role for Sulf1 in the ventral neural tube, and suggests a mechanism whereby Sulf1 activity shapes the Shh morphogen gradient by promoting ventral accumulation of high levels of Shh protein.

Sulf1 influences the Shh morphogen gradient during the dorsal ventral patterning of the neural tube in Xenopus tropicalis.

The organization and maturation of ventricular cardiomyocytes from the embryonic to the adult form is crucial for normal cardiac function. We have shown that a polarity protein, Scrib, may be involved in regulating the early stages of this process. Our goal was to establish whether Scrib plays a cell autonomous role in the ventricular myocardium, and whether this involves well-known polarity pathways.

Scrib:Rac1 interactions are required for the morphogenesis of the ventricular myocardium.

Planar cell polarity (PCP) is the mechanism by which cells orient themselves in the plane of an epithelium or during directed cell migration, and is regulated by a highly conserved signalling pathway. Mutations in the PCP gene Vangl2, as well as in other key components of the pathway, cause a spectrum of cardiac outflow tract defects. However, it is unclear why cells within the mesodermal heart tissue require PCP signalling. Using a new conditionally floxed allele we show that Vangl2 is required solely within the second heart field (SHF) to direct normal outflow tract lengthening, a process that is required for septation and normal alignment of the aorta and pulmonary trunk with the ventricular chambers. Analysis of a range of markers of polarised epithelial tissues showed that in the normal heart, undifferentiated SHF cells move from the dorsal pericardial wall into the distal outflow tract where they acquire an epithelial phenotype, before moving proximally where they differentiate into cardiomyocytes. Thus there is a transition zone in the distal outflow tract where SHF cells become more polarised, turn off progenitor markers and start to differentiate to cardiomyocytes. Membrane-bound Vangl2 marks the proximal extent of this transition zone and in the absence of Vangl2, the SHF-derived cells are abnormally polarised and disorganised. The consequent thickening, rather than lengthening, of the outflow wall leads to a shortened outflow tract. Premature down regulation of the SHF-progenitor marker Isl1 in the mutants, and accompanied premature differentiation to cardiomyocytes, suggests that the organisation of the cells within the transition zone is important for maintaining the undifferentiated phenotype. Thus, Vangl2-regulated polarisation and subsequent acquisition of an epithelial phenotype is essential to lengthen the tubular outflow vessel, a process that is essential for on-going cardiac morphogenesis.

Simon A. Ramsbottom

Institute of Genetic Medicine

Newcastle University

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

Simon A. Ramsbottom

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Genetic Medicine

Newcastle University

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

Institute of Genetic Medicine