Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.
???displayArticle.pubmedLink???
26709854
???displayArticle.pmcLink???PMC4694024 ???displayArticle.link???J Vis Exp ???displayArticle.grants???[+]
Briscoe,
Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling.
1999, Pubmed
Briscoe,
Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling.
1999,
Pubmed
Chamberlain,
Notochord-derived Shh concentrates in close association with the apically positioned basal body in neural target cells and forms a dynamic gradient during neural patterning.
2008,
Pubmed
Chen,
The zebrafish Nodal signal Squint functions as a morphogen.
2001,
Pubmed
Christian,
Xwnt-8, a Xenopus Wnt-1/int-1-related gene responsive to mesoderm-inducing growth factors, may play a role in ventral mesodermal patterning during embryogenesis.
1991,
Pubmed
,
Xenbase
Clevers,
Wnt/beta-catenin signaling in development and disease.
2006,
Pubmed
Dessaud,
Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network.
2008,
Pubmed
Du,
Identification of distinct classes and functional domains of Wnts through expression of wild-type and chimeric proteins in Xenopus embryos.
1995,
Pubmed
,
Xenbase
Erickson,
Formation and maintenance of morphogen gradients: an essential role for the endomembrane system in Drosophila melanogaster wing development.
2011,
Pubmed
Fay,
Wilcoxon-Mann-Whitney or t-test? On assumptions for hypothesis tests and multiple interpretations of decision rules.
2010,
Pubmed
Fellgett,
Sulf1 has ligand-dependent effects on canonical and non-canonical Wnt signalling.
2015,
Pubmed
,
Xenbase
Freeman,
Extracellular regulation of developmental cell signaling by XtSulf1.
2008,
Pubmed
,
Xenbase
Ingham,
Hedgehog signaling in animal development: paradigms and principles.
2001,
Pubmed
Janda,
Structural basis of Wnt recognition by Frizzled.
2012,
Pubmed
,
Xenbase
McDowell,
Activin has direct long-range signalling activity and can form a concentration gradient by diffusion.
1997,
Pubmed
,
Xenbase
Miller,
Establishment of the dorsal-ventral axis in Xenopus embryos coincides with the dorsal enrichment of dishevelled that is dependent on cortical rotation.
1999,
Pubmed
,
Xenbase
Müller,
Differential diffusivity of Nodal and Lefty underlies a reaction-diffusion patterning system.
2012,
Pubmed
Ramsbottom,
Sulf1 influences the Shh morphogen gradient during the dorsal ventral patterning of the neural tube in Xenopus tropicalis.
2014,
Pubmed
,
Xenbase
Ribes,
Distinct Sonic Hedgehog signaling dynamics specify floor plate and ventral neuronal progenitors in the vertebrate neural tube.
2010,
Pubmed
Rushlow,
Temporal dynamics, spatial range, and transcriptional interpretation of the Dorsal morphogen gradient.
2012,
Pubmed
Schohl,
Beta-catenin, MAPK and Smad signaling during early Xenopus development.
2002,
Pubmed
,
Xenbase
Shen,
Nodal signaling: developmental roles and regulation.
2007,
Pubmed
Shimmi,
New insights into extracellular and post-translational regulation of TGF-β family signalling pathways.
2013,
Pubmed
Tada,
Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway.
2000,
Pubmed
,
Xenbase
Tao,
Maternal wnt11 activates the canonical wnt signaling pathway required for axis formation in Xenopus embryos.
2005,
Pubmed
,
Xenbase
Towers,
Gradients of signalling in the developing limb.
2012,
Pubmed
Williams,
Visualizing long-range movement of the morphogen Xnr2 in the Xenopus embryo.
2004,
Pubmed
,
Xenbase
Wolpert,
One hundred years of positional information.
1996,
Pubmed
,
Xenbase
Yu,
Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules.
2009,
Pubmed
