This work has received funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation program (grant agreement n&#176; 759282).

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(65), 34290 (2012).</li><li>Grossmann, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RootChip:+an+integrated+microfluidic+chip+for+plant+science.">The RootChip: an integrated microfluidic chip for plant science.</a> Plant Cell. 23 (12), 4234-4240 (2011).</li><li>Brunoud, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+sensor+to+map+auxin+response+and+distribution+at+high+spatio-temporal+resolution.">A novel sensor to map auxin response and distribution at high spatio-temporal resolution.</a> Nature. 482, 103-106 (2012).</li><li>Wang, N. N., Shin, M. C., Li, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GUS+reporter-aided+analysis+of+the+promoter+activities+of+Arabidopsis+ACC+synthase+genes+AtACS4,+AtACS5,+and+AtACS7+induced+by+hormones+and+stresses.">The GUS reporter-aided analysis of the promoter activities of Arabidopsis ACC synthase genes AtACS4, AtACS5, and AtACS7 induced by hormones and stresses.</a> Journal of Experimental Botany. 56 (413), 909-920 (2005).</li><li>Alabadi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gibberellins+Repress+Photomorphogenesis+in+Darkness.">Gibberellins Repress Photomorphogenesis in Darkness.</a> Plant Physiology. 134 (3), 1050-1057 (2004).</li><li>De Lucas, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+framework+for+light+and+gibberellin+control+of+cell+elongation.">A molecular framework for light and gibberellin control of cell elongation.</a> Nature. 451 (7177), 480-484 (2008).</li><li>Strasser, B., Sanchez-Lamas, M., Yanovsky, M. 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The authors have nothing to disclose.

The FRET-based GA biosensor nlsGPS1 provides a quantitative method to report and measure GA hormone gradients in multicellular plants. FRET-based biosensors can quantify dynamics with an improved spatiotemporal resolution over direct detection by mass spectrometry and indirect measurement by transcriptional reporters or signaling-protein-degradation-based methods12,13. High-resolution cellular imaging in diverse tissue-types can yield meaningful insights into GA biology and spark new hypotheses regarding the regulation and function of GA accumulations in a multicellular context. For example, monitoring changes in the nlsGPS1 biosensor in specific GA biosynthetic, catabolic, and transport mutants, as well as during spatiotemporally induced perturbations, could be very informative to test specifically how GA gradients are established in the root and address root cell responses to GA gradients. The sensor could be used in other model and crop species to test the conservation of the mechanisms that control the GA-mediated control of seed germination, cellular elongation, and flowering.
The critical steps in the FRET-based imaging of the nlsGPS1 biosensor are that, 1) the pixels should not be saturated during the quantitative FRET analysis, 2) imaging parameters such as &#34;detector gain&#34; should be kept constant for the donor emission (DxDm) and acceptor emission (DxAm) acquisitions, 3) control nlsGPS1-NR lines should be used to rule out artefacts, and 4) samples should be prepared to minimize drift and focal-change issues. Additionally, the environmental conditions in which samples are grown are important to control since GA levels are sensitive to environmental conditions such as light duration and light intensity14,15,16,17. A key limitation of this type of analysis is that a high signal-to-noise ratio is required for imaging due to the increase in noise inherent in ratiometric imaging. Thus, nlsGPS1 imaging will not be useful for tissues and organs that are not amenable to ratiometric fluorescence microscopy using cyan and yellow fluorescent proteins&#8212;for example, deeper tissues where fluorescent proteins are poorly detected. On the other hand, ratiometric readouts are often preferred over intensiometric readouts, because an internal control is helpful to rule out artefacts stemming from changes in biosensor expression, stability, brightness, or detectability in a given cell, tissue, or condition. For example, FRET biosensor imaging and image analyses have also been used to study a variety of ligands in a variety of tissues5,6,18,19,20,. The imaging experiments and image analyses reported here can be modified to suit new imaging methods, such as light sheet microscopy, that could yield novel insights in, for example, deeper root tissue-types.
The first-generation nlsGPS1 biosensor is a high-affinity sensor that provides a high-resolution map of GA gradients that can also report on intracellular increases in GA following exogenous GA treatments. One of the current limitations of nlsGPS1 is that the sensor is not rapidly reversible and, thus, reports not on steady-state GA levels but, likely, on the maximum recent GA concentration in the solution of interest. The precise turnover rate for the sensor is also not known and this, combined with low reversibility, precludes detection of endogenous GA depletions that might be happening within a minutes to few a hours in some tissue-types. It is also important to note that nlsGPS1 has a high affinity for GA4 (Kd = 24 nM) compared to other GA forms (GA3&#160;Kd= 240 nM, GA1&#160;Kd = 110 nM) when imaging other bioactive GAs7. Future generations of GA biosensors can be engineered to increase reversibility while maintaining high affinity or to exhibit different specificities for the various precursor, bioactive, or catabolite GAs.

Plant hormones play a fundamental role in plant growth and development. These small, mobile signaling molecules are typically regulated at several levels, such as biosynthesis, catabolism, and short- and long-distance transport1,2,3,4. The understanding of hormone signaling pathways and downstream transcriptional responses has sharpened over the years. However, to link the diverse cellular responses of the hormone signaling pathways with the regulatory inputs directing hormone distributions, we require a spatiotemporal quantification of hormone levels at a cellular scale. FRET-based biosensors that can detect phytohormones can advance scientists&#39; ability to quantify hormone levels at a cellular scale. FRET-based biosensors consist of a FRET pair (donor and acceptor fluorescent proteins) linked to a sensory domain that binds a specific ligand or responds to a biological stimulus. For small molecule biosensors, ligand binding triggers a conformational change of the sensory domain that results in a change of distance and/or orientation between the two fluorescent proteins of the FRET pair. A ratiometric analysis of a FRET biosensor is accomplished by exciting the donor and measuring the fluorescence emission ratio of acceptor over donor5,6. Ligand binding is detectable as a change in this emission ratio7.
We recently developed a FRET-based biosensor for the plant hormone GA. GAs are a class of hormones that can promote seed germination, cellular elongation, and the developmental transition from vegetative to flowering phases. The nlsGPS1 biosensor is nuclear localized and provides spatiotemporal insights into GA dynamics in diverse plant tissues. In Arabidopsis cells, GA binds to soluble receptors, gibberellin-insensitive dwarf (GID), and the complex induces the degradation of DELLA proteins that act as negative regulators of GA signalling2. The GA sensory domain of nlsGPS1 consists of the Arabidopsis GA receptor (AtGID1C) linked to a 74-amino acid truncation of a DELLA protein (AtGAI) and a FRET pair consisting of enhanced dimerization variants of Cerulean as the donor fluorescent protein and Aphrodite (a codon-diversified Venus) as the acceptor fluorescent protein8. The nlsGPS1 biosensor is a high-affinity sensor for the bioactive GA4 (Kd = 24 nM for GA4) and it can be utilized in diverse tissue-types to map and quantify GA gradients. To avoid misinterpretation of the Arabidopsis GA levels in vivo, we have also developed a nonresponsive variant of nlsGPS1 (nlsGPS1-NR) to use as a negative control. The nlsGPS1-NR protein carries mutations in the GA-binding pocket that disrupt the binding of GA and mutations in the DELLA protein that disrupt the interaction with GID receptor proteins7,9. Emission ratio patterns or changes observed in both nlsGPS1 and nlsGPS1-NR lines can be considered artefacts not directly related to GA-binding events. It is also important to note that nlsGPS1 binding to GA4 is not rapidly reversible, and therefore, cellular nlsGPS1 emission ratios should be interpreted as representing the highest recent concentration of GA in a given nucleus rather than the real-time steady-state levels. As a consequence, an analysis of falling GA levels is not possible with nlsGPS1.
Here we provide a detailed protocol for utilizing a nlsGPS1 biosensor in cells of the model plant Arabidopsis, using confocal imaging-based approaches at a high-resolution. The protocol provides information on imaging plant roots and hypocotyls both at steady state and over time-courses. The nlsGPS1 sensor could potentially be utilized in diverse tissue-types, as well as across plant species, to map and quantify GA distributions.

<table>
	<tbody>
		<tr>
			<td>nlsGPS1 Col0 Arabidopsis seeds</td>
			<td>NASC</td>
			<td>N2107734</td>
			<td></td>
		</tr>
		<tr>
			<td>nlsGPS-NR Col0 Arabidopsis seeds</td>
			<td>NASC</td>
			<td>N2107735</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibberellin A4 (GA4)</td>
			<td>Sigma</td>
			<td>G7276</td>
			<td>dissolve in EtH70 % , and keep at -20&#176;C</td>
		</tr>
		<tr>
			<td>sodium hypochlorite solution (Bleach)</td>
			<td>Fisher S/5040</td>
			<td>HSRA 064</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen cloride HCl</td>
			<td>Sigma</td>
			<td>31434</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropore tape</td>
			<td>3M</td>
			<td>1530-1</td>
			<td></td>
		</tr>
		<tr>
			<td>ibidi sticky-slide</td>
			<td>Ibidi</td>
			<td>81128</td>
			<td>Luer 0.1 for root imaging</td>
		</tr>
		<tr>
			<td>ibidi sticky-slide</td>
			<td>Ibidi</td>
			<td>80168</td>
			<td>Luer 0.2 for hypocotyl imaging</td>
		</tr>
		<tr>
			<td>glass coverslip for sticky slides</td>
			<td>Ibidi</td>
			<td>10812</td>
			<td></td>
		</tr>
		<tr>
			<td>Elbow Luer Connectors</td>
			<td>Ibidi</td>
			<td>10802</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone tubing</td>
			<td>Ibidi</td>
			<td>108401</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock Connector</td>
			<td>Ibidi</td>
			<td>10826</td>
			<td></td>
		</tr>
		<tr>
			<td>programmable syringe pump</td>
			<td>World Precision Instruments</td>
			<td>AL-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum grease</td>
			<td>Sigma</td>
			<td>18405</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige and Skoog Basal Salts</td>
			<td>Duchefa</td>
			<td>M0221</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar plant, 1kg</td>
			<td>Melford</td>
			<td>P1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide ground edges, 76mm x 26mm, 1.0mm to 1.2mm thick</td>
			<td>Fisher Scientific</td>
			<td>12383118</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slip No.1 1/2 glass 22mm x 22mm</td>
			<td>Fisher Scientific</td>
			<td>12363138</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-slip Syringe 2o ml</td>
			<td>Fisher Scientific</td>
			<td>10785126</td>
			<td></td>
		</tr>
		<tr>
			<td>3M Micropor Surgical Paper Tape</td>
			<td>Fisher Scientific</td>
			<td>12787597</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hydroxide, 500g</td>
			<td>Sigma Aldrich</td>
			<td>221473-500G-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute Ethanol</td>
			<td>Fisher Scientific</td>
			<td>10428671</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Watchmaker 5 StSteel</td>
			<td>Scientific Laboratory Supplies</td>
			<td>INS4340</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, 125mm, stainless steel</td>
			<td>Fisher Scientific</td>
			<td>12338099</td>
			<td></td>
		</tr>
		<tr>
			<td>Fitting reducer 0.5 to 1.6</td>
			<td>Ibidi</td>
			<td>10829</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8</td>
			<td></td>
			<td></td>
			<td>Confocal laser microscope 1</td>
		</tr>
		<tr>
			<td>Zeiss LSM 780</td>
			<td></td>
			<td></td>
			<td>Confocal laser microscope 2</td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td>3D visualization and Analysis software</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td></td>
			<td>image analysis software</td>
		</tr>
		<tr>
			<td>OriginPro</td>
			<td>Origin Lab</td>
			<td></td>
			<td>Statistical Analysis Software</td>
		</tr>
	</tbody>
</table>

visualizing cellular gibberellin levels using the nlsgps1 f&#246;rster resonance energy transfer (fret) biosensor

The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental transitions in plants. Gibberellin Perception Sensor 1 (GPS1) is the first F&#246;rster resonance energy transfer (FRET)-based biosensor that allows monitoring of cellular GA levels in vivo. By measuring a fluorescence emission ratio of nuclear localized-GPS1 (nlsGPS1), spatiotemporal mapping of endogenously and exogenously supplied GA gradients in different tissue types is feasible at a cellular scale. This protocol will describe how to image nlsGPS1 emission ratios in three example experiments: steady-state, before-and-after exogenous gibberellin A4 (GA4) treatments, and over a treatment time-course. We also provide methods to analyze nlsGPS1 emission ratios using both Fiji and a commercial three-dimensional (3-D) micrograph visualization and analysis software and explain the limitations and likely pitfalls of using nlsGPS1 to quantify gibberellin levels.

Using nlsGPS1, it is possible to measure cellular GA4 levels in tissues amenable to fluorescence imaging, including root tips and dark-grown hypocotyls (Figure 2). In the Arabidopsis root, the nlsGPS1 emission ratio gradient is indicative of low GA levels in the meristematic and division zones and high GA levels in the late elongation zone (Figure&#160;2A and 2B). In contrast, an emission ratio gradient was not observed in nlsGPS1-NR roots, suggesting that the endogenous GA gradient is not an artefact (Figure&#160;2C and 2D).A nlsGPS1 emission ratio gradient was also formed in dark-grown hypocotyls, with low levels in the cotyledons and the apical hook and high levels in the rapidly elongating basal region of the hypocotyl (Figure&#160;2E and 2F). In contrast, an emission ratio gradient was not observed in the nlsGPS1-NR hypocotyls (Figure&#160;2G and 2H). In both Arabidopsis roots and dark-grown hypocotyl cells, endogenous GA accumulation correlated with cellular elongation rate.
Furthermore, exogenously supplied GA4 accumulates preferentially in the elongation zone compared to the division zone of the Arabidopsis root (Figure 3), indicating that nlsGPS1 can be used to study endogenous and exogenous GA patterning.
During time course experiments, nlsGPS1 seedlings were placed in sticky-slide chambers and perfused with &#188; MS liquid, followed by a treatment with 0.1 &#181;M GA4 for 30 min. The video shows a faster accumulation of exogenous GA4 in the root elongation zone compared to the division zone (Video 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58739/58739fig1.jpg" /> 
Figure 1: Sample preparation for confocal imaging. These panels show a schematic representation of the sample preparation for (A) a steady-state experiment, (B) before-and-after exogenous GA4 treatments, and for (C) a treatment time course experiment using sticky-slides (C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58739/58739fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58739/58739fig2.jpg" /> 
Figure 2: The GA gradient in Arabidopsis roots and dark-grown hypocotyls. Two-dimensional images of (A) nlsGPS1 and (C) nlsGPS1-NR roots were analyzed using ImageJ software, and three-dimensional images of (B) nlsGPS1 and (D) nlsGPS1-NR were analyzed using a commercial three-dimensional image analysis software. Both analyses showed an endogenous GA4 gradient in Arabidopsis roots. Two-dimensional images of (E) nlsGPS1 and (G) nlsGPS1-NR dark-grown hypocotyl were analyzed using ImageJ software, and three-dimensional images of (F) nlsGPS1 and (H) nlsGPS1-NR were analyzed using the commercial three-dimensional image analysis software. Both analyses showed an endogenous GA4 gradient in dark-grown hypocotyls. The LUT bar displays the false coloration of nlsGPS1 emission ratios. YFP images are reported as expression controls. Hypocotyl images were acquired using two stage positions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58739/58739fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58739/58739fig3.jpg" /> 
Figure 3: The exogenous GA gradient in roots. The first two panels show (A) two-dimensional and (B) three-dimensional images of a nlsGPS1 root before and 20 min after the treatment of exogenous GA4 (1 &#181;M). YFP images are reported as expression controls. The last two panels show (C) the mean and standard deviation and (D) beeswarm and box plot of nlsGPS1 emission ratios for nuclei of the elongation zone (the region which is defined with a white frame). In the elongation zone, the nlsGPS1 emission ratio was significantly higher after GA4 treatment (Mann-Whitney U test, *** P-value &#60; 0.0001). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58739/58739fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1" src="/files/ftp_upload/58739/58739movie1.jpg" /> 
Video 1: Perfusion experiment of nlsGPS1 root using sticky-slide. This video shows three-dimensional images of nlsGPS1 perfused with &#188; MS liquid and treated with 0.1 &#181;M GA4 for 30 min. In the time course, imaging was acquired every 10 min for 3 h with the following intervals: 30 min of mock solution (frame t = 1, t = 2, t = 3), 30 min of GA4 treatment (frame t = 4, t = 5, t = 6), 2 h of mock solution (frame t = 7 to t = 18) solution. Prior to the acquisition, the sample was perfused with mock solution for 2 h. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58739/Movie1.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Visualizing Cellular Gibberellin Levels Using the nlsGPS1 FÃƒÂ¶rster Resonance Energy Transfer FRET Biosensor at JoVE.com

Visualizing Cellular Gibberellin Levels Using the nlsGPS1 F&#246;rster Resonance Energy Transfer (FRET) Biosensor

Gibberellin Perception Sensor 1 (GPS1) is the first F&#246;rster resonance energy transfer-based biosensor for measuring the cellular levels of gibberellin phytohormones with a high spatiotemporal resolution. This protocol reports on the method to visualize and quantify cellular gibberellin levels using the genetically encoded nlsGPS1 biosensor in Arabidopsis hypocotyls and root tips.

The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental ...

visualizing-cellular-gibberellin-levels-using-nlsgps1-frster

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.6K Views.  University of Cambridge. Gibberellin Perception Sensor 1 (GPS1) is the first Förster resonance energy transfer-based biosensor for measuring the cellular levels of gibberellin phytohormones with a high spatiotemporal resolution. This protocol reports on the method to visualize and quantify cellular gibberellin levels using the genetically encoded nlsGPS1 biosensor in Arabidopsis hypocotyls and root tips.

Video: Visualizing Cellular Gibberellin Levels Using the nlsGPS1 Förster Resonance Energy Transfer (FRET) Biosensor

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

The interrenal gland in zebrafish is the teleostean counterpart of the mammalian adrenal gland. This protocol introduces how to perform the 3-&#946;-hydroxysteroid dehydrogenase (&#916;5-4 isomerase; 3&#946;-Hsd) enzymatic activity assay, which detects differentiated steroidogenic cells in the developing zebrafish.

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...