This work was funded by a BBSRC grant to MEP (BB/H010297/1), a BBSRC quota studentship to SAR, and an MRC studentship to SWF.

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The authors have no competing financial interests.

An essential part of this protocol is generating biologically active ligands that are normally processed, secreted, and able to elicit a response in the receiving cell, despite having a fluorescent moiety attached. It is critical to establish that the fluorescently-tagged gene product is biologically active using an appropriate assay. For Shh-GFP, the ability to activate the expression of ptc1 was confirmed16. Wnt8a has a remarkably potent ability to induce a secondary axis when expressed in a single ventral blastomere20-21, and Wnt8a-HA-eGFP was shown retain this biological activity. Wnt11b inhibits activin treated ectodermal explants from undergoing convergent extension21-22, and Wnt11b-HA-eGFP also retains its biological activity17. The ability of the tagged ligands to elicit responses equivalent to the untagged proteins suggests that the conclusions based on experiments using the fluorescent ligands will be relevant to the normal protein. The addition of the HA epitope between the ligand and the fluorescent protein provided a spacer region that allows the Wnt constructs to be both active and fluorescent.
The major limitation of this type of analysis is that it does not directly inform on endogenous morphogen gradients. For instance, the diffusion of Shh across the field of cells in animal cap may not reflect the way endogenous Shh moves through the columnar neural epithelium in the embryo. However, the simplicity of this protocol allows experiments where the effect of exogenous regulators on the distribution of ligands can be tested. The results from these approaches can form the basis of hypotheses to be tested using more demanding in vivo analyses. For instance, the ability of over-expressed Sulf1 to restrict the distribution of Shh-GFP using the methods described in this paper pointed to the potential for Sulf1 in modulating the Shh morphogen gradient. This was directly tested and validated using antisense morpholino knock-down of Sulf1 and immunocytochemistry to visualise endogenous Shh in the neural tube16. A similar method to ours has been used to investigate specific mechanisms that could regulate ligand diffusion. Smith and colleagues demonstrated that a GFP-tagged nodal (Xnr2) used simple diffusion, and not cell extensions (cytonemes) or transcytosis (using vesicles) to form a gradient23.
Further elaborations of these methods are possible where other proteins that block particular pathways can be expressed in targeted cells to investigate whether a response to the signalling molecule is needed as an intermediary to relay signals from one cell to another. For instance, expressing a dominant negative activin receptor between the source of activin and the responding cells showed that simple ligand diffusion could elicit a response several cell diameters away from the source even with non-responsive cells in between24. This conclusion was supported by work in zebrafish where wild type cells can respond to a source of nodal, despite being surrounded by non-responsive mutant cells lacking an essential co-receptor for nodal25. Other studies have used zebrafish to investigate diffusion of fluorescently tagged ligands28,29. Some studies has observed that epitope tagging the C-terminus of Wnt proteins can impact on signalling activity, possibly because of the highly conserved cysteines that contribute to protein conformation. Our previous work has shown that including a spacer (such as the HA tag) results in tagged Wnt ligands that are biologically active17. This is likely because the spacer provides flexibility needed for ligand &#8211;receptor interaction, such as in the thumb-forefinger model of Wnt-Frz binding30.&#160;
The next step for advancing our studies is to include a visual readout for the activation of the signalling pathway. For instance, expressing GFP-tagged Dvl26 in cells distant from the source of ligand will allow the range across which Wnt11b has ability to signal to be measured. The range of Wnt8a signalling activity could be shown using antibody staining to measure nuclear localisation of b-catenin in cells27 at a distance from the source. These types of experiments are possible using the techniques described in this paper. By employing a variety of different fluorescent proteins or fluorophores, an additional level of information would be provided where one not only measures the distribution of a ligand but the also the output of the signalling pathways, and in this way determining the threshold range of a morphogen.
Xenopus laevis is a useful model for visualising GFP-tagged ligands and further details on the methods for procuring embryos, mRNA synthesis, microinjection and dissection are available31.

During early embryonic development, cells are progressively committed to follow specific lineages of differentiation: this means a group of totipotent (or pluripotent) cells become gradually restricted to establishing populations of progenitor cells determined to give rise to one cell type. Cell-cell signalling is central to the regulation of lineage specification during embryonic development. Manipulation of these signals will be required to direct stem cells toward particular fates to support novel medical treatments.
A relatively small number of signalling pathways are reiterated during development, including pathways responding to the TGF superfamily (nodals and BMPs)1-2, FGFs3, Wnts4, and Hedgehogs5. These secreted proteins bind receptors present on the cell membrane to activate signal transduction thereby altering gene expression and/or cell behaviour. The tight regulation of cell signalling is essential for cell lineage specification and normal development. While the cross-talk among these pathways is important in determining cell fate, a single ligand can itself elicit distinct responses at different concentrations. Morphogen gradients were described over 100 years ago as a theory to explain how different cell types can derive from a field of cells6. Signalling molecules produced by one group of cells may diffuse over a certain range, decreasing in concentration with a greater distance from the source. Cells exposed to the signal will respond to the local concentration at their position in the field of cells, with cells at distinct positions responding differently to different levels of the signal. Evidence for the existence of morphogens comes from studies of the early Drosophila embryo7 and the wing disc8, as well as the vertebrate limb9 and neural tube10.
Methods are needed to investigate how morphogen gradients are established and to identify other molecules important in regulating these gradients. Elegant experiments using immunohistochemistry to visualise endogenous proteins in vivo in the context of different genetic backgrounds have been used to investigate morphogen gradients11-12. However, good antibodies and specific mutants are not always available, so we describe here a protocol using overexpression of fluorescent ligands in Xenopus, to provide an alternative, simple method to dissect how exogenous gene products can influence distribution of ligands across a field of cells. Xenopus laevis provides an excellent system to undertake these types of experiments as their embryos develop externally so they are accessible at the earliest stages. Their large size (1-1.5mm in diameter) simplifies microinjection and surgical manipulation and by blastula stages the cells are easy to image as they are still relatively big (about 20&#181;m across). Overexpression studies in Xenopus are simple to do: mRNA injected into the early embryo can be targeted to particular cells and is efficiently translated.
The fluorescently tagged Wnt8a/Wnt11b-HA-eGFP constructs were generated using pCS2 Wnt8a-HA13, pCS2 Wnt11b-HA14 and eGFP. The HA peptide is important to include, not only to provide an additional molecular tag, but also because it is thought to act as a spacer separating the Wnt and eGFP proteins allowing both gene products to function. The construct used for the visualisation of Shh was previously used to generate a transgenic mouse expressing a Shh-eGFP fusion protein15; this was kindly provided by Andy McMahon. Importantly, the GFP tag for all the constructs is cloned 3&#8217; to the signal sequence such that it is retained after processing. It is also essential to ensure that the final protein includes sequences required for modifications, such the addition of lipids as is the case for Shh and Wnt ligands.The cDNAs were subcloned into the pCS2+ expression vector which is optimised for the prodution of synthetic mRNA; it includes an SP6 promoter and polyadenylation signal (http://sitemaker.umich.edu/dlturner.vectors).
The work described here provides a simple protocol for comparing the secretion and diffusion of fluorescently tagged Wnt and Shh tagged ligands. By injecting defined amounts of synthetic mRNA, the protocol circumnavigates any problems associated with variable expression from different vectors using different promoters. These methods have recently been applied to investigate the effects of the heparan sulfate endosulfatase Sulf1 on Shh-eGFP and Wnt8a/Wnt11b-HA-eGFP secretion and diffusion in Xenopus16-17.

<table><tbody><tr><td>Agarose</td><td>Melford</td><td>MB 1200</td><td></td></tr><tr><td>Ammonium acetate</td><td>Ambion</td><td>From Megascript SP6 Kit AM 1330</td><td></td></tr><tr><td>Bicarbonate</td><td>VWR International</td><td>RC-091</td><td></td></tr><tr><td>Calcium nitrate</td><td>Sigma</td><td>C13961</td><td></td></tr><tr><td>Cap analog (m7G(5&#039;))</td><td>Applied Biosystems</td><td>AM 8050</td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>C 2432</td><td></td></tr><tr><td>L-Cysteine hydrochloride monohydrate</td><td>Sigma</td><td>C7880</td><td></td></tr><tr><td>dNTPs</td><td>Invitrogen</td><td>18427-013</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma</td><td>O3690</td><td></td></tr><tr><td>Ethanol</td><td>VWR International</td><td>20821.33</td><td></td></tr><tr><td>Ficoll 400</td><td>Sigma</td><td>F 4375</td><td></td></tr><tr><td>Fiji image J software</td><td></td><td></td><td>Free download http://fiji.sc/Fiji</td></tr><tr><td>Gentamycin</td><td>Melford</td><td>G 0124</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Fisher Scientific</td><td>A/0400/PB17</td><td></td></tr><tr><td>Glass cover slips, No.1.5&amp;nbsp;</td><td>Scientific Laboratory Supplies</td><td>22X22-SGJ3015. 22X50-SGJ3030&amp;nbsp;</td><td></td></tr><tr><td>Glass needle puller</td><td>Narishige</td><td>Narishige PC -10</td><td></td></tr><tr><td>Glass pull needles</td><td>Drummond Scientific</td><td>3-000-203-G/X</td><td></td></tr><tr><td>Human chronic gonadotropin (HCG)</td><td>Intervet</td><td></td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>P/7500/PB17</td><td></td></tr><tr><td>Lithium chloride (LiCl)</td><td>Sigma</td><td>L-7026</td><td></td></tr><tr><td>LSM710 and Zen software (2008-2010)</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Matlab software</td><td>Mathworks</td><td></td><td>http://uk.mathworks.com/</td></tr><tr><td>Molecular grade water&amp;nbsp;</td><td>Fisher Scientific</td><td>BP 2819-10</td><td></td></tr><tr><td>Nail varnish&amp;nbsp;</td><td>Boots</td><td>Bar code 3600530 373048</td><td></td></tr><tr><td>Spectrophotometer</td><td>Lab.tech International</td><td>ND-1000 / ND8000</td><td></td></tr><tr><td>Petri dish (55 mm)</td><td>VWR International</td><td>391-0865</td><td></td></tr><tr><td>Phenol-chloroform</td><td>Sigma</td><td>P3803</td><td></td></tr><tr><td>Photoshop software</td><td>Adobe</td><td></td><td>http://www.photoshop.com/products</td></tr><tr><td>High fidelity DNA polymerase and buffers&amp;nbsp;</td><td>Biolabs</td><td>M0530S&amp;nbsp; Buffer - M0531S</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Fisher Scientific</td><td>P/4280/53</td><td></td></tr><tr><td>PVC insulation tape</td><td>Onecall</td><td>SH5006MPK</td><td></td></tr><tr><td>Gel extraction kit&amp;nbsp;</td><td>Qiagen</td><td>S28704</td><td></td></tr><tr><td>Restriction enzymes buffers</td><td>Roche</td><td>SuRE/CUT Buffer Set 11082 035 001</td><td></td></tr><tr><td>RNAse-free DNAse</td><td>Promega</td><td>ME10A</td><td></td></tr><tr><td>Steel back single edge blades</td><td>Personna</td><td>66-0403-0000</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher Scientific</td><td>27810.364</td><td></td></tr><tr><td>SP6 transcription kit</td><td>Ambion</td><td>AM1330</td><td></td></tr><tr><td>Glass slides</td><td>Thermo Fisher</td><td>SHE 2505</td><td></td></tr><tr><td>Tris base</td><td>Invitrogen</td><td>15504-020</td><td></td></tr><tr><td>Tungsten needles</td><td></td><td></td><td>homemade</td></tr><tr><td>Zen lite software</td><td>Carl Zeiss</td><td></td><td>Free download&amp;nbsp; http://www.zeiss.co.uk/microscopy/en_gb/downloads/zen.html</td></tr></tbody></table>

using confocal analysis of xenopus laevis 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 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.

Confocal analysis of animal cap explants expressing fluorescently tagged proteins provides an effective system for visualising ligand distribution under different experimental conditions. In one example, the distribution of GFP tagged Shh is shown (Figure 1). At the 2-cell stage, Xenopus embryos are injected into both cells with either with a control mRNA or with mRNA coding for Sulf1, an enzyme that modifies cell surface heparan sulfate and influences the Shh morphogen gradient16. These embryos are cultured until the 32-cell stage and a single cell is co-injected with mRNAs coding for Shh-GFP and memRFP (as a lineage tracer). This creates a clone of cells expressing Shh-GFP that is marked with the cell membrane tethered RFP. At the blastula stage, animal cap explants are taken for analysis by confocal microscopy. Figure 1 shows that under control conditions, Shh-GFP is secreted and diffuses outside of the region expressing the injected mRNAs. However, in the presence of Sulf1, Shh-GFP is more restricted in its distribution and, in this sample, is not detected outside the clone of cells producing it. The effects of Sulf1 on Shh-GFP distribution has been analysed more fully16.
When expressed in Xenopus embryos, fluorescently tagged Wnt ligands are secreted, accumulate on the cell membrane, and diffuse across the field of cells. In Figure 2, we characterise the quantitaive and qualitative properties of the two different fluorecently tagged Wnt ligands in cells injected and expressing the proteins. Figure 2A-H shows examples of how Wnt8a and Wnt11b-HA-eGFP accumulate on the membrane of animal cap cells. By comparing panels 2B and 2F it is clear that Wnt8a-HA-eGFP accumulates more efficiently on the cell membrane than Wnt11b-HA-eGFP. Quantitative information has been extracted from the confocal images using a combination of image and script analysis software (supplemental code file). Figure 2I shows the relative accumulation of Wnt8a-HA-eGFP on the cell membrane compared to Wnt11b-HA-eGFP. The data was obtained using a computer script that determines the total number of Wnt-HA-eGFP pixels co-localising with cell membrane pixels in each image. In another approach, the total number of Wnt-HA-eGFP puncta that co-localise with the cell membrane are counted using image analysis software (Figure 2J). Qualitative information about the size and shape of individual puncta can also be extracted from the data using image analysis software. In addition to fewer Wnt11b-HA-eGFP puncta co-localising with the cell membrane (Figure 2J) these puncta also have a smaller average size than Wnt8a-HA-eGFP puncta (Figure 2K). Moreover, Wnt11b-HA-eGFP puncta have a reduced circularity compared to Wnt8a-HA-eGFP puncta (Figure 2L). Circularity is a measure of how closely an object resembles a perfect circle, with 1 representing a perfect circle and 0.1 an elongated non-circular shape. This approach can be used to test any candidate regulator of Wnt ligand diffusion. To do this, control cells should be injected with a control mRNA coding for an inactive form of the candidate regulator or an irrelevant protein such as beta-galactosidase (lacZ); this provides a more valid control than simply not injecting the regulator. Data in these examples was analysed using the non-parametric test Mann-Whitney U18-19. A statistical analysis programme was used to perform the test.
In Figure 3, we investigate Wnt ligand diffusion. A single blastomere was injected at the 4-cell stage such that the animal cap explants represent a field of cells in which only some cells express either Wnt8a-HA-eGFP or Wnt11b-HA-eGFP. By including a cell lineage tracer, we can identify expressing versus non-expressing cells and this allows the range of Wnt8a-HA-eGFP and Wnt11b-HA-eGFP ligand diffusion away from the source cells to be analysed (Figure 3B and 3C). Due to the curvature of the animal cap explants, the maximum distance that could be reliably measured using this assay was 160&#181;m, which was not enough to measure the absolute distance of ligand diffusion. However, by using a combination of confocal analysis, image analysis and scientific analysis and graphing software the overall shape of the Wnt-HA-eGFP morphogen gradient could be analysed (Figure 3D). This data can be used to investigate whether overexpressing another protein together with the tagged ligands in the same cells can affect Wnt secretion or diffusion. In another type of experiment (Figure 3E-3J) the effects of a potential regulator in receiving cells can be measured by overexpressing the candidate protein in clones of cells adjacent to cells expressing Wnt8a or Wnt11b-HA-eGFP. This allows any non-cell-autonomous effects of the regulator on Wnt-HA-eGFP ligand diffusion to be examined. Consistent with Figure 2, more Wnt8a-HA-eGFP can be detected diffusing away from cells than Wnt11b-HA-eGFP in both diffusion experiments.
The types of experiments described in this paper have been used to analyse the effect of Sulf1 on Shh and Wnt signalling16,17.
<img alt="Figure 1" src="/files/ftp_upload/53162/53162fig1.jpg" /> 
Figure 1. Modulation of Shh-GFP distribution can be detected by confocal analysis of Xenopus animal caps. (A) A cartoon depicting experimental assay where embryos are first injected with mRNA coding for Sulf1 or a control mRNA, the same embryos are later injected at the 32-cell stage with mRNA coding for Shh-GFP into a single cell. (B-C) Animal cap explants were taken at stage 8 and imaged after 4 hr. Images are shown at the edge of a Shh-GFP + memRFP expressing clone of cells. In control embryos (B), Shh-GFP is distributed away from the memRFP marked clone of signal producing cells. In embryos expressing Sulf1 (C), Shh-GFP is more restricted in its distribution, see16. memRFP is shown in magenta and Shh-GFP in green. Scale bars represent 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53162/53162fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53162/53162fig2.jpg" /> 
Figure 2.&#160;Quantitative and qualitative analysis of Wnt8a and Wnt11b-HA-eGFP puncta on the cell membrane. (A-H) Embryos were microinjected bi-laterally with mRNA encoding memRFP (500 pg) into the animal hemisphere at the two cell stage. In addition embryos were injected with mRNA encoding (A-D) Wnt8a-HA-eGFP (500 pg) or [E-H] Wnt11b-HA-eGFP (1ng). The embryos were also injected with mRNA coding for LacZ to provide a control for additional mRNA/protein when analysing the effects of any potential regulator. The white boxes in (C) and (G) mark the areas enlarged in panels (D) and (H) respectively. The total amount Wnt-HA-eGFP fluorescence co-localising with the cell membrane was calculated using image and script analysis software and then normalised (I). Qualitative information was extracted using image analysis software. Wnt8a and Wnt11b-HA-eGFP punctae were analysed for particle number (J), particle size (K) and particle circularity (L). Mann-Whitney U (**P&#60;0.01), N=number of embryos. memRFP is shown in magenta, and Wnt8a/11b-HA-GFP is shown in green, scale bars represent 20&#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53162/53162fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53162/53162fig3.jpg" /> 
Figure 3.&#160;Measuring the range of diffusion of fluorescently tagged Wnt ligands. (A) Diagram depicting the assay used to measure Wnt8a and Wnt11b-HA-eGFP secretion and diffusion away from expressing cells. (B-C) mRNA encoding (B) memCerulean (600 pg), LacZ (4ng) and Wnt8a-HA-eGFP (2ng) or (C) memCerulean (600 pg), LacZ (4ng) and Wnt11b-HA-eGFP (2 ng) was injected into the animal hemisphere of one blastomere at the four cell stage. (D) The range of diffusion of Wnt8a-HA-eGFP and Wnt11b-HA-eGFP was measured through a control background. (E) Diagram depicting the assay used to measure Wnt8a and Wnt11b-HA-eGFP diffusion through a background expressing LacZ, see method for details. (F-G) mRNA encoding memCerulean (600 pg) and (F and H) Wnt8a-HA-eGFP (2 ng) or (G and I) Wnt11b-HA-eGFP was injected into the animal hemisphere of one blastomere at the four cell stage. An adjacent blastomere was injected with mRNA encoding memRFP (600 pg) and LacZ (4 ng) see Key for details. (J) The range of Wnt8a-HA-eGFP and Wnt11b-HA-eGFP diffusion was measured through a background expressing LacZ. The data was quantified and plotted using confocal analysis, image analysis and scientific analysis and graphing software. memCerulean (blue (C-D) and Yellow (F and H)), Wnt-HA-eGFP (green), memRFP (magenta), scale bars represent 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53162/53162fig3large.jpg" target="_blank">Please click here to download a larger version of this file.</a>
<img alt="Table 1" src="/files/ftp_upload/53162/53162table1.jpg" />
Table 1. Primers used to subclone Wnt8a/Wnt11b-HA-eGFP and Shh-GFP into pCS2.
<table>
	<tbody>
		<tr>
			<td>Reaction</td>
			<td>Components</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Example CS2+ digest</td>
			<td>1.5 &#956;g of pCS2+</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 &#956;l of Restriction enzyme 1</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 &#956;l of Restriction enzyme 2</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>5 &#956;l of Restriction enzyme buffer (10X)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Made up to 50 &#956;l with molecular grade water</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Example mRNA synthesis</td>
			<td>2 &#956;l linearised template</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 &#956;l 10x Megascript trx mix</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 &#956;l 50mM ATP</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 &#956;l 50mM CTP</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 &#956;l 50mM UTP</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 &#956;l 5mM GTP</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2.5 &#956;l 40mM Cap Analog (m7G(5&#39;)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 &#956;l SP6 enzyme mix</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>3.5 &#956;l Molecular grade water</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Example PCR reaction</td>
			<td>0.5 &#956;l High fidelity DNA polymerase (2,000 units/ml)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 ng Template DNA</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2.5 &#956;l of forward and reverse primers (10&#956;M)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>0.5 &#956;l of dNTPs</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>5 &#956;l of DNA ploymerase buffer (10X)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Made up to 50 &#956;l with molecular grade water</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Example PCR conditions</td>
			<td>Intial denaturation 2 min at 98 &#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>15 sec 98 &#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>15 sec 65 &#176;C</td>
			<td>30 cycles</td>
		</tr>
		<tr>
			<td></td>
			<td>40 sec 72 &#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Final extension 10 min at 72 &#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Example PCR product digest</td>
			<td>28 &#956;l of PCR product</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 &#956;l of Restriction enzyme 1</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 &#956;l of Restriction enzyme 2</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>5 &#956;l of Restriction enzyme buffer (10X)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>13 &#956;l Molecular grade water</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Example T4 ligation</td>
			<td>1 &#956;l Cut CS2+</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>3 &#956;l Cut PCR product 1</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>3 &#956;l Cut PCR product 2</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1 &#956;l of T4 DNA ligase</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1 &#956;l T4 DNA ligase buffer (10X)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1 &#956;l Molecular grade water</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Example template digest</td>
			<td>5 ug Plasmid DNA</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>10 &#956;l Restriction enzyme buffer (10X)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>3 &#956;l Not1 (except Shh-GFP, Kpn1 is used)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Made up to 100 &#956;l with molecular grade water</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2. Example reaction conditions used for subcloning and producing synthetic mRNA for Wnt8a-HA-eGFP. 
<table>
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Components</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cysteine-HCL</td>
			<td>0.1X NAM</td>
		</tr>
		<tr>
			<td></td>
			<td>2.5% L-cysteine hydrochloride monohydrate (pH7.8)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NAM salts</td>
			<td>110 mM NaCL</td>
		</tr>
		<tr>
			<td></td>
			<td>2 mM KCl</td>
		</tr>
		<tr>
			<td></td>
			<td>1 mM CA(NO3)2</td>
		</tr>
		<tr>
			<td></td>
			<td>0.1 mM EDTA</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NAM/2</td>
			<td>0.5X NAM salts</td>
		</tr>
		<tr>
			<td></td>
			<td>5 mM HEPES pH7.4</td>
		</tr>
		<tr>
			<td></td>
			<td>0.25 mM Bicarbonate</td>
		</tr>
		<tr>
			<td></td>
			<td>25 ug/ml Gentamycin</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NAM/3 + Ficoll</td>
			<td>0.33X NAM salts</td>
		</tr>
		<tr>
			<td></td>
			<td>5 mM HEPES pH7.4</td>
		</tr>
		<tr>
			<td></td>
			<td>0.25 mM Bicarbonate</td>
		</tr>
		<tr>
			<td></td>
			<td>2 5ug/ml Gentamycin</td>
		</tr>
		<tr>
			<td></td>
			<td>5 % Ficoll</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NAM/10</td>
			<td>0.1X NAM salts</td>
		</tr>
		<tr>
			<td></td>
			<td>5 mM HEPES pH7.4</td>
		</tr>
		<tr>
			<td></td>
			<td>25 ug/ml Gentamycin</td>
		</tr>
	</tbody>
</table>
Table 3. Solutions used during the production and microinjection of Xenopus laevis embryos.

Watch this Scientific Journal Video about Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients at JoVE.com

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

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

Using Confocal Analysis of Xenopus laevis 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 ...

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11.1K Views.  The Bateson Centre, University of Sheffield. The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

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

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Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

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Technique to Target Microinjection to the Developing Xenopus Kidney

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Technique to Target Microinjection to the Developing Xenopus Kidney

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Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

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The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

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Neuroscience

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Biology

Flow Cytometry and Confocal Imaging Analysis of Low Wnt Expression in Axin2-mTurquoise2 Reporter Thymocytes

Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt signaling operates via a dosage-driven mechanism, motivating the need to study and measure Wnt signaling in various cell types. Although several reporter models have been proposed to represent physiological Wnt expression, either the genetic context or the reporter protein highly influenced the validity, accuracy, and flexibility of these tools. This paper describes methods for acquiring and analyzing data obtained with the Axin2-mTurquoise2 mouse Wnt reporter model, which contains a mutated Axin2em1Fstl allele. This model facilitates the study of endogenous canonical Wnt signaling in individual cells over a wide range of Wnt activity.
This protocol describes how to fully appreciate Axin2-mTurquoise2 reporter activity using cell population analysis of the hematopoietic system, combined with cell surface markers or &#946;-catenin intracellular staining. These procedures serve as a base for implementation and reproduction in other tissues or cells of interest. By combining fluorescence-activated cell sorting and confocal imaging, distinct canonical Wnt expression levels can be visualized. The recommended measurement and analysis strategies provide quantitative data on the fluorescent expression levels for precise assessment of canonical Wnt signaling. These methods will be useful for researchers who want to use the Axin2-mTurquise2 model for canonical Wnt expression patterns.

Signaling levels are known to regulate cell fate, indicating that regulation of Wnt signaling constitutes an interesting therapeutic target. Here, we describe flow cytometry and confocal microscopy analysis methods for a robust murine canonical Wnt signaling reporter model that measures distinct Wnt signaling levels.

Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt ...

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

Summary

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Chapters in this video

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Technique to Target Microinjection to the Developing Xenopus Kidney

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Neural Explant Cultures from Xenopus laevis

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Flow Cytometry and Confocal Imaging Analysis of Low Wnt Expression in Axin2-mTurquoise2 Reporter Thymocytes