We thank Yumiko Iwamoto for supporting the production of the video. We are also grateful to Akihiro Isomura for discussion and supports of image analysis, Hitoshi Miyachi for technical supports for generation of transgenic animals, Yuji Shinjo (Olympus Medical Science), Masatoshi Egawa (Olympus Medical Science), Takuya Ishizu (Olympus Medical Science) and Ouin Kunitaki (Andor Japan) for the technical support and discussions of the bioluminescence imaging system. This work was supported by Core Research for Evolutional Science and Technology (JPMJCR12W2) (R.K.), Grant-in-Aid for Scientific Research on Innovative Areas (MEXT 24116705 for H.S. and MEXT 16H06480 for R.K.), Grant-in-Aid for Scientific Research (C) (JSPS 18K06254) (H.S.), Takeda Foundation (R.K. and H.S.), and Platform for Dynamic Approaches to Living System from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

All the procedure including animal subjects have been approved by Institutional Animal Care and Use Committee at the institute for Frontier Life and Medical Sciences, Kyoto University.
1. Bioluminescence reporters
NOTE: The luciferase reporter is suitable for measuring the rapid dynamics of promoter activity by fusing the degradation signal. Moreover, the luciferase fusion reporter enables monitoring of the protein dynamics in the single cell. Both types of reporters are available for mono-layer culture (dissociation culture) and tissue culture (slice culture) experiment.
<ol>
	<li>Reporter for monitoring Dll1 promoter activity17 
	NOTE: To monitor the rapid changes in gene expression, the rapid-response and unstable reporter is essential. Firefly luciferase (Fluc) shows rapid maturation compared with fluorescein reporters. Because ubiquitin fused luciferase reporter (Ub-Fluc) shows rapid degradation and rapid-turnover, it is very useful to monitor the dynamic gene expression in living cells20.
	<ol>
		<li>Use Dll1 promoter-driven destabilized luciferase reporter, ubiquitinated luciferase (pDll1-Ub-Fluc, Figure 1A upper panel), to monitor rapid changes in Dll1 expression at a transcriptional level17.</li>
		<li>Generate a transgenic mouse line carrying pDll1-Ub-Fluc for the stable expression of this reporter.</li>
	</ol>
	</li>
	<li>Reporter for monitoring Dll1 protein dynamics17.
	<ol>
		<li>For generation of knock-in mice, insert the luciferase cDNA into the 3&#180; termini of the Dll1 coding region so that Dll1-luciferase fusion protein is expressed (Dll1-Fluc reporter, Figure 1A, lower panel)17.</li>
		<li>Monitor the expression dynamics of Dll1 at protein levels by measuring the luciferase activity.</li>
		<li>For monitoring Dll1 protein dynamics at tissue levels, introduce the Dll1-Fluc reporter into NPCs in the embryonic telencephalon by in utero electroporation.</li>
	</ol>
	</li>
</ol>
2. Bioluminescence imaging system
<ol>
	<li>Construct a bioluminescence live-imaging system using an inverted microscope installed with a highly sensitivity, water-cooled CCD camera. In order to reduce the noise and obtain high sensitivity, cool the camera with chilled water provided by the water circulator at an optimized temperature of -90 &#176;C.</li>
	<li>For the live cell/tissue imaging, install the incubation system, controlling the temperature and mixed gas, to the microscope stage.</li>
	<li>For imaging the expression of destabilized luciferase reporter, use high numerical angle (NA: 1.30) 40x oil-immersion objective lens. The working distance of this lens is 0.2 mm, it is available for not only mono-layer cell culture but also slice culture.</li>
	<li>For dual monitoring of luciferase and fluorescein reporter expression, install the LED illumination device to the light path of the microscope.</li>
	<li>Control the time-lapse imaging with a software associated with the microscope (e.g., Multidimensional acquisition program of MetaMorph software).</li>
	<li>Prepare the entire system in a dark room, because the signal of the destabilized luciferase reporter is very faint, and to avoid the extraneous light prevent the acquisition.</li>
</ol>
3. Neural Stem/Progenitor Cell (NPC) dissociation cultures
<ol>
	<li>Preparation of culture media, dishes, and reagents for NPC dissociation culture
	<ol>
		<li>Prepare N2/B27 media for culturing neural stem/progenitor cells with a final concentration of 1x N2, 1x B27, 1 mM N-acetylcysteine, 10 ng/mL bFGF and 50 U/mL Penicillin/Streptomycin in DMEM/F12 media.</li>
		<li>Prepare papain solution for dissociation containing 7 U/mL papain, 0.006% DNase and 1 mM N-acetylcysteine in EBSS media.</li>
		<li>Prepare 100 mM of luciferin sodium solution for luminescence imaging. Dilute 100 mM luciferin solution in N2/B27 media to a final concentration of 1 mM. 
		NOTE: Luciferin shows auto-fluorescein itself. In the case of luciferase-fluorescence dual imaging, especially EGFP, lowering the concentration of luciferin to 0.5 mM is better.</li>
		<li>Coat a 35-mm glass bottom dishes (glass diameter 27-mm) with 40 &#956;g/mL of poly-L-lysine (PLL) solution for 1 h at room temperature. After incubation, wash the plates 3x with PBS and let it dry.</li>
	</ol>
	</li>
	<li>Dissection of embryos and dissociation of NPCs
	<ol>
		<li>After euthanizing a pregnant pDll1-Ub-luc transgenic mouse (embryonic day 12.5 (E12.5)) by CO2 asphyxiation and cervical dislocation, cut through the abdomen and take out the uterus. 
		NOTE: Researchers must follow the regulations and guidelines of animal research committee of their institution. If anesthesia or pain relief drugs are used, use them properly.</li>
		<li>Transfer the uterus in a 10 cm Petri dish containing 25 mL of ice-cold PBS. Take out the embryos from the uterus in ice-cold PBS, using micro scissors and fine forceps.</li>
		<li>Cut off the head of each embryo using scissors and transfer it to 10 cm Petri dishes containing ice-cold DMEM/F12. Remove the epidermis and cartilage surrounding the brain and transfer the brain to 35 mm Petri dish containing ice-cold 3 mL of N2/B27 media.</li>
		<li>Cut off the right and left telencephalon from diencephalon (Figure 1Ba-f, C,D). Remove the meninges covering the surface of telencephalon using fine forceps (Figure 1Bi). Using fine forceps again, take out the dorso-lateral part of cortex from the telencephalon (Figure 1Bg,h,j-l E).</li>
		<li>Transfer the tissues to new 1.5 mL tubes using a P1000 pipette and remove any extra medium with a P200 pipette. Add 0.1 mL of papain solution per brain tissue (a pair of the cortex). After 15 min of incubation at 24 &#176;C, gently pipette the samples 10 times with P1000 pipette. Incubate the samples again for 15 min at 24 &#176;C.</li>
		<li>Gently pipette the samples 10 times with P1000 pipette. Centrifuge the samples for 3 min at 400 x g and room temperature (RT). Discard the supernatant.</li>
		<li>Add 1 mL of DMEM/F12 media to the pellet. Gently pipette 10 times with P1000 pipette. Centrifuge the samples for 3 min at 400 x g and RT. Discard the supernatant. Repeat this at least 2 more times.</li>
		<li>To the pellet, add 0.5 mL of N2/B27 media containing 1 mM luciferin and mix well. Seed the cells (1 x 106 cells) to a PLL-coated glass bottom dish. Culture the cells in the CO2 incubator for 1 h. Once the cells adhere to the dish, add 2 mL of N2/B27 media, containing 1 mM luciferin.</li>
	</ol>
	</li>
	<li>Visualization of luciferase reporter expression in NPC dissociation culture
	<ol>
		<li>Start up the luminescence live-imaging system before performing the dissection. For NPC culture, set the temperature of the stage incubator at 37 &#176;C and set the setting of the gas mixture 20% O2, 5% CO2.</li>
		<li>Put the immersion oil to the objective lens. Place the sample dish on the microscope stage. View the field manually and choose the best position and the focus of the cells of interest. Click live to acquire the test image.</li>
		<li>Run the time-lapse acquisition by 2-dimensional (luminescence and bright field) acquisitions for 24 h with Multi-Dimensional Acquisition program. Choose Multi-Dimensional Acquisition program and set the acquisition setting as follows (step 3.3.6).</li>
		<li>For luminescence image acquisition, use the following camera settings: low-transfer rate (50 kHz), 2 x 2/4 x 4 binning, 10 min/5 min exposure time. For bright-field image acquisition, use the following settings: intermediate-transfer rate (1 MHz), 1 x 1 binning and 100 ms exposure time.</li>
	</ol>
	</li>
</ol>
4. In utero electroporation
NOTE: This is performed for the introduction of Dll1-Fluc reporter into the neural progenitor cells.
<ol>
	<li>Preparation of tools and reagents for electroporation
	<ol>
		<li>Prepare micro capillaries for injection of DNA into the ventricle of the embryonic telencephalon. Stretch a grass capillary with heat and cut and polish the tip of it with the polishing machine.</li>
		<li>Prepare a mixture of DNA with dye (e.g., Trypan blue). The concentration of each DNA is 1 &#956;g/&#956;L in PBS and add the dye to a final concentration of 10%. 
		NOTE: Use the mixture of DNA for visualization of Dll1 protein dynamics as follows: Dll1-Fluc (Dll1 protein reporter, Figure 2E upper) and pEF-EGFP (EF promoter driven EGFP expression vector, Figure 2E lower), monitoring the location and morphology of transfected cells.</li>
		<li>Prepare two types of anesthetics: 3.88 mg/mL of pentobarbital and 0.12% of xylazine, in sterile 1x PBS.</li>
		<li>Use the sterile instruments and surgical gloves during operation.</li>
	</ol>
	</li>
	<li>In utero electroporation
	<ol>
		<li>Anesthetize a time pregnant ICR mouse (E12.5) by intraperitoneal administration of anesthetics with 500 &#956;L of 3.88 mg/mL pentobarbital and 500 &#956;L of 0.12% xylazine. 
		NOTE: Researchers must follow the regulation of animal experiments. If one needs to use anesthesia or pain relief drugs, use them properly.</li>
		<li>Clip the hair and disinfect the skin with surgical scrub.</li>
		<li>Check for the lack of toe pinch response. Once pedal reflex is not observed, make a 2-3 cm cut through the abdomen along the midline. Put gauzes around the dissected part and wet the gauzes with warm PBS not to dry up the incision. Take out the right uterine horn gently with ring-shaped forceps and count the number of embryos.</li>
		<li>Inject 1-2 &#956;L of the mixed DNA into the ventricle of the telencephalon of embryos gently with micro capillary. 
		NOTE: When the injection is successful, one can see the blue color of the dye over the surface of the uterus.</li>
		<li>Wet the uterus and the electrode before providing the voltage pulses, and then hold the head of an embryo gently. Set the positively charged electrode to the side of the hemisphere, in which the DNA was injected. Ensure that the condition of the pulse of electroporation is 30-50 V for 50 ms, the interval of the pulses is 1 s (50 ms charge and 950 ms non-charge). Provide 5 pulses to one embryo and check that bubbles are generated from the negatively charged electrode. 
		NOTE: Make sure the direction of the electrode: DNA is incorporated into cells on the side of positive electrode. During this procedure, do not move the position of the electrode.</li>
		<li>Repeat the steps 4.2.3-4.2.4 for other embryos and return the uterine horn to the abdominal cavity. Carry out the same procedure to the embryos in the left uterine horn.</li>
		<li>After putting back the left side, suture the incision by a silk suture (4-0) with a needle (17 mm). Before closing the incision completely, put warm PBS into the abdominal cavity 
		NOTE: The interval between sutures is around 2 mm.</li>
		<li>After finishing the surgery, place the mouse on a heating pad for post anesthesia recovery. House the mouse individually. 
		NOTE: Researchers must follow their local instiutional regulation of animal experiments. If one needs to use pain relief drugs, use them properly.</li>
	</ol>
	</li>
</ol>
5. Preparation of slice cultures of the developing cortex and visualization of luciferase reporter expression in the cortical slices
<ol>
	<li>Preparation of culture media and reagents for slice cultures of the cortex
	<ol>
		<li>Prepare enriched media for culturing cortical slices containing 5% fetal bovine serum and 5% horse serum in N2/B27 media.</li>
		<li>Bubble 100% O2 gas through DMEM/F12 media for dissection and making cortical slices. 
		NOTE: To use oxygen gas safely, store the O2 cylinder in the cylinder cabinet and monitor the oxygen concentration in the air by an oxygen concentration meter.</li>
		<li>Dilute 100 mM luciferin sodium solution in enriched media to a final concentration of 1 mM. 
		NOTE: Luciferin shows auto-fluorescein itself. In the case of luciferase-fluorescence dual imaging, especially EGFP, lower concentration of luciferin (0.5 mM) is better.</li>
		<li>Set the multi-gas incubator at 37 &#176;C by 40% O2 and 5% CO2.</li>
	</ol>
	</li>
	<li>Dissection of embryos and examination of the expression of fluorescent reporter
	<ol>
		<li>After euthanizing the pregnant mouse to which the reporters (E13.5) are introduced by in utero electroporation (step 4.2), cut through the abdomen and take out the uterus. 
		NOTE: Researchers must follow the regulation of animal experiments. If using anesthesia or pain relief drugs, use them properly.</li>
		<li>Transfer the uterus to a 10 cm Petri dish containing PBS. Take out the embryos from uterus in PBS, using micro scissors and fine forceps. Cut off the head of each embryo and transfer to a 10 cm Petri dish containing DMEM/F12 media bubbled with 100% O2 gas. Remove epidermis and cartilage surrounding the brain in DMEM/F12 media.</li>
		<li>Put the brain on the lid of the Petri dish with ring-shaped forceps and set the lid on the fluorescence stereoscopic microscope stage. Check the region of the cortex expressing fluorescent protein under the excitation light (Figure 2A,B).</li>
		<li>Transfer the brain to a silicone rubber cutting board filled with 30 mL of DMEM/F12 media bubbled with 100% O2 gas. Remove meninges covering the surface of the telencephalon by fine forceps.</li>
		<li>Cut the border between medial and lateral part of the dorsal telencephalon and separate into two hemispheres using micro surgical knife or fine forceps. Using a micro surgical knife, cut the cortex like stripes and make cortical slices (Figure 2C,D). Pipette slices with medium using pipette and transfer them to enriched media in a 35 mm dish.</li>
		<li>Put the slices on the culture inserts in the glass bottom dishes with enriched media. Correct the direction of slices using fine forceps. Set up the cut surface of the slices to the surface of culture insert. Remove the extra media by a pipette. Incubate the slices in multi-gas incubator set by 40% O2 and 5% CO2 at 37 &#176;C for 30 min.</li>
		<li>Add 300 &#956;L of enriched media containing 1 mM luciferin to the outside of culture insert in the glass bottom dish.</li>
	</ol>
	</li>
	<li>Visualization of luciferase reporter expression in slice cultures
	<ol>
		<li>Start the luminescence live-imaging system before starting the dissection. Use the following condition of cortical slice cultures: 40% O2, 5% CO2 and 37 &#176;C.</li>
		<li>Put the immersion oil to 40x objective lens. Put the sample dish to the stage of microscope. Acquire the test image of fluorescent and set the position and the focus plane to the region of interest under the illumination of excitation light.</li>
		<li>Run the time-lapse acquisition by 3-dimentional (luminescence, fluorescence and bright field) acquisitions for 24 h (Figure 2F-H). For luminescence image acquisition, use the following camera settings: low-transfer rate (50 kHz), 2x2/4x4 binning, 10 min/5 min exposure time. For the fluorescence and bright field image acquisition, use the following settings: intermediate-transfer rate (1 MHz), 1x1 binning and 100 ms exposure time.</li>
	</ol>
	</li>
</ol>
6. Image processing and analysis
<ol>
	<li>Connect each image and make the stack images. Click Import Image Sequence from the File menu (File | Import | Image Sequence) and choose the folder, where the acquired images are saved. Put some words contained in file name to the column of File name contains.</li>
	<li>To remove the noise of cosmic ray on the bioluminescence images, apply the SpikeNoise Filter plug-in of ImageJ/Fiji. Open the stack images and click SpikeNoise Filter.</li>
	<li>Apply Savitzky Golay Temporal Filter plug-in to get clear dynamics of reporter expression. Open the stack images and click Savitzky Golay Temporal Filter.</li>
	<li>To measure the intensity of reporter expression, apply Z Axis Profile Plus plug-in to each single cell. Select the cells and set ROIs, region of interest and click Z Axis Profile Plus.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+Gene+Expression+in+a+Single+Cell.">Stochastic Gene Expression in a Single Cell.</a> Science. 297, 1183-1186 (2002).</li><li>Nelson, D. E., 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=Oscillations+in+NF-kappaB+signaling+control+the+dynamics+of+gene+expression.">Oscillations in NF-kappaB signaling control the dynamics of gene expression.</a> Science. 306, 704-708 (2004).</li><li>Purvis, J. E., 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=p53+dynamics+control+cell+fate.">p53 dynamics control cell fate.</a> Science. 336, 1440-1444 (2012).</li><li>Hansen, A. S., O'Shea, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoter+decoding+of+transcription+factor+dynamics+involves+a+trade-off+between+noise+and+control+of+gene+expression.">Promoter decoding of transcription factor dynamics involves a trade-off between noise and control of gene expression.</a> Molecular Systems Biology. 9, 704 (2014).</li><li>Hansen, A. S., O'Shea, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cis+Determinants+of+Promoter+Threshold+and+Activation+Timescale.">cis Determinants of Promoter Threshold and Activation Timescale.</a> Cell Reports. 12, 1226-1233 (2015).</li><li>Johnson, H. E., Toettcher, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+Dynamics+Control+Cell+Fate+in+the+Early+Drosophila+Embryo.">Signaling Dynamics Control Cell Fate in the Early Drosophila Embryo.</a> Developmental Cell. 48, 361-370 (2019).</li><li>Badr, C. E., Tannous, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging:+Progress+and+applications.">Bioluminescence imaging: Progress and applications.</a> Trends in Biotechnology. 29, 624-633 (2011).</li><li>Nakajima, Y., Ohmiya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+assays:+multicolor+luciferase+assay,+secreted+luciferase+assay+and+imaging+luciferase+assay.">Bioluminescence assays: multicolor luciferase assay, secreted luciferase assay and imaging luciferase assay.</a> Expert Opinion on Drug Discovery. 5, 835-849 (2010).</li><li>Nakajima, Y., 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=Enhanced+beetle+luciferase+for+high-resolution+bioluminescence+imaging.">Enhanced beetle luciferase for high-resolution bioluminescence imaging.</a> PLoS One. 5, (2010).</li><li>Stacer, A. C., 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=NanoLuc+reporter+for+dual+luciferase+imaging+in+living+animals.">NanoLuc reporter for dual luciferase imaging in living animals.</a> Molecular Imaging. 12, (2013).</li></ol>

The authors have no conflicting financial interest.

The components of Notch signaling show oscillatory expressions in synchrony during somitogenesis but out of synchrony during neurogenesis, leading to the difficulties in capturing the expression dynamics by static analysis in the latter case. Thus, real-time monitoring is required to reveal the expression dynamics of Notch signaling components, such as Hes1 and Dll1. Because the periods of the expressions of Hes1 and Dll1 oscillations are extremely short, approximately 2-3 h, rapid res...

The mammalian brain is composed of a large number of various types of neurons and glial cells. All cells are generated from neural stem/progenitor cells (NPCs), which first proliferate to expand their numbers, then start to differentiate into neurons, and finally give rise to glial cells1,2,3,4,5. Once cells have differentiated into neurons, they cannot proliferate or increase their numbers, and, therefore, the maintenance of NPCs until later stages is important. Notch signaling via cell-cell interactions plays an important role in maintaining NPCs6,7. Notch ligands interact with the membrane protein, Notch, on the surface of neighboring cells and activates the Notch protein. After activation, proteolysis of Notch protein occurs, thereby releasing the intracellular domain of Notch (NICD) from the cell membrane into the nucleus8,9,10. In the nucleus, NICD binds to the promoter regions of Hes1 and Hes5 (Hes1/5) and activates the expression of these genes. Hes1/5 repress the expression of the proneural genes Ascl1 and Neurogenin1/2 (Neurog1/2)11,12,13,14. Because proneural genes induce neuronal differentiation, Hes1/5 play essential roles in maintaining NPCs. Furthermore, as proneural genes can activate the expression of the Notch ligand Delta-like1 (Dll1), Hes1/5 also repress the expression of Dll1. Therefore, the expression of Dll1 leads to neighboring cells being negative for Dll1 via Notch signaling. In this way, cells inhibit adjacent cells from following their same fate, a phenomenon known as the lateral inhibition8. In the developing brain, lateral inhibition plays a role in generating various different cell types.
Real-time imaging at the single cell level reveals dynamic expressions of the components of Notch signaling in NPCs15,16,17. Notch signaling activates the expression of Hes1, but Hes1 protein binds to its own promoter and represses its own expression. Furthermore, Hes1 is an extremely unstable protein, that is degraded by the ubiquitin-proteasome pathway; therefore, the repression of its own promoter is only short lived and then the transcription starts again. In this way, the expression of Hes1 oscillates at both the transcription and translational levels in a 2 h cycle18. The oscillatory expression of Hes1, in turn, induces the oscillatory expression of the downstream target genes, such as Ascl1, Neurog2, and Dll1, via periodic repression15,16,17,19. While proneural genes can induce neuronal differentiation, their oscillatory expression is not sufficient for neuronal differentiation; rather their sustained expression is essential for the neuronal differentiation. The oscillatory expression of proneural genes is important for maintaining NPCs rather than for inducing neuronal differentiation14,15,16. The expression of Dll1 oscillates at both the transcription and translational levels during various morphogenesis, such as neurogenesis and somitogenesis. The dynamic expression of Dll1 is important for the normal morphogenesis and steady expression of Dll1 induces defects in neurogenesis and somitogenesis17. These findings demonstrate the important function that the dynamics of gene expression and protein kinetics have on the regulation of various developmental events (i.e., different expression dynamics produce different outputs in cellular behaviors).
To analyze the dynamics of Notch signaling, the static analysis of tissues and cells are insufficient because they are constantly changing. Real-time imaging of single cells is a powerful tool to reveal the dynamics in gene expression. The dynamic expression of Notch signaling molecules undergo rapid cyclic responses in the period of 2-3 h. This rapid periodic expression presents two difficult problems for the real-time monitoring: (1) the expression of the molecules is suppressed to low levels, and (2) rapid turnover requires fast-response reporters. To overcome these problems, we previously developed a bioluminescence real-time imaging method20. Because the bioluminescence reporter has a higher sensitivity and shorter maturation time than fluorescent reporters, this strategy enables us to monitor the rapid dynamics in living cells. Using real-time visualization, we found that more genes exhibited dynamic expression than we had previously thought. In addition, the number of reports showing expression and protein dynamics in living cells and the significance of these dynamics in various biological events has increased, suggesting a fundamental role of the dynamics in gene expressions21,22.
In this report, we describe a way to visualize the expression of the Notch ligand Dll1 in NPCs in both dissociated cultures and in cortical slice cultures. To monitor the dynamics of Dll1 transcription at single cell levels, we generated dissociated cultures of NPCs derived from the embryonic telencephalon of transgenic mice carrying pDll1-Ub-Fluc reporter, a Dll1 promoter-driven destabilized luciferase reporter. To monitor Dll1 protein dynamics in vivo, we introduced the Dll1-Fluc fusion reporter into NPCs in the cortex and visualized the expression of the reporter in NPCs in cortical slice cultures. Real-time imaging enabled us to capture the various features of gene expression and protein dynamics in living cells at high temporal resolution.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bioluminescence Imaging System</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chilled water circulator (chiller)</td>
			<td>Julabo</td>
			<td>Model: F12-ED</td>
			<td></td>
		</tr>
		<tr>
			<td>Cooled CCD camera</td>
			<td>Andor Technology</td>
			<td>Model: iKon-M 934</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator system</td>
			<td>TOKAI HIT</td>
			<td>Model: INU-ONICS</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>Model: IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>Model: IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>LED illumination device</td>
			<td>CoolLED</td>
			<td>Model: pE1</td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph</td>
			<td>MOLECULAR DEVICES</td>
			<td>Model: 40000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mix gas controller</td>
			<td>Tokken</td>
			<td>Model: TK-MIGM OLO2</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Olympus</td>
			<td>Model: UPLFLN 40X O</td>
			<td></td>
		</tr>
		<tr>
			<td>Preparations for Dissection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>Model: SMZ-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence stereoscopic microscope</td>
			<td>Leica</td>
			<td>Model: MZ16FA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>DUMONT</td>
			<td>INOX No.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, Micro scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ring-shaped forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10-cm plastic petri dish</td>
			<td>greiner</td>
			<td>664160-013</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm plastic petri dish</td>
			<td>greiner</td>
			<td>627160</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Nacalai Tesque</td>
			<td>14249-24</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>invitrogen</td>
			<td>11039-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents for NPC dissociation culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>invitrogen</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>invitrogen</td>
			<td>13256-029</td>
			<td>Stock solution: 1 &#956;g/ml in 0.1% BSA/PBS</td>
		</tr>
		<tr>
			<td>D-luciferin</td>
			<td>Nacalai Tesque</td>
			<td>01493-85</td>
			<td>Stock solution: 100mM in 0.9% saline</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003172</td>
			<td>Stock solution: 1000U/ml in EBSS</td>
		</tr>
		<tr>
			<td>EBSS</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003188</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dish</td>
			<td>IWAKI</td>
			<td>3910-035</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement (100x)</td>
			<td>invitrogen</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-cystein</td>
			<td>Sigma</td>
			<td>A-9165-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003178</td>
			<td>Stock solution: 7U/ml in EBSS</td>
		</tr>
		<tr>
			<td>Penicillin/Streptmycine</td>
			<td>Nacalai Tesque</td>
			<td>09367-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma</td>
			<td>P-6281</td>
			<td>40 mg/ml in DW</td>
		</tr>
		<tr>
			<td>Preparations for in utero electroporation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50-ml syringe</td>
			<td>TERUMO</td>
			<td>181228T</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode</td>
			<td>Neppagene</td>
			<td>7-mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Neppagene</td>
			<td>CUY21 EDIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gauzes</td>
			<td>Kawamoto co.</td>
			<td>7161</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro capillary</td>
			<td>Made in-house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Nacalai Tesque</td>
			<td>14249-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentbarbital</td>
			<td>Kyoritsuseiyaku</td>
			<td>Somnopentyl</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring-shaped forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, Micro scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suture needle</td>
			<td>Akiyama MEDICAL MFG. CO</td>
			<td>F17-40B2</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>Seractal</td>
			<td></td>
		</tr>
		<tr>
			<td>Preparations for Slice culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10-cm plastic petri dish</td>
			<td>greiner</td>
			<td>664160-013</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm plastic petri dish</td>
			<td>greiner</td>
			<td>627160</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture insert</td>
			<td>Millipore</td>
			<td>PICM01250</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>invitrogen</td>
			<td>11039-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>172012-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>DUMONT</td>
			<td>INOX No.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro surgical knife</td>
			<td>Alcon</td>
			<td>19 Gauge V-Lance</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-gas incubator</td>
			<td>Panasonic</td>
			<td>MCO-5MUV-PJ</td>
			<td></td>
		</tr>
		<tr>
			<td>N2/B27 media</td>
			<td>Made in-house</td>
			<td></td>
			<td>ref. NPC dissociatioin culture</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Nacalai Tesque</td>
			<td>14249-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring-shaped forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, Micro scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon rubber cutting board</td>
			<td>Made in-house</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time bioluminescence imaging of notch signaling dynamics during murine neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Expressions of the genes Hes1/7 exhibit 2 h oscillation cycle in various cell lines and during somitogenesis. Furthermore, the period of oscillation is very short and both their mRNAs and proteins are extremely unstable with the half-lives of around 20 min. If using a slow response reporter, we cannot trace such rapid dynamics, and if using a stable reporter, it gradually accumulates while the gene expression oscillates. Thus, the reporter must be rapidly degraded to monitor the rapid turnover of such cyclically...

Watch this Scientific Journal Video about Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis at JoVE.com

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

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7.5K Views.  Kyoto University. Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Video: Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Studying Wnt Signaling During Patterning of Conducting Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. 
This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing organs of both invertebrates and vertebrates, asymmetrically dividing progenitors generates a Notchhi self-renewing and a Notchlo differentiating daughter. In the embryonic zebrafish brain, radial glia progenitors (RGPs)-the principal vertebrate neural stem cells-mostly undergo ACD to give birth to one RGP and one differentiating neuron. The optical clarity and easy accessibility of zebrafish embryos make them ideal for in vivo time-lapse imaging to directly visualize how and when the asymmetry of Notch signaling is established during ACD. Recent studies have shown that dynamic endocytosis of the Notch ligand DeltaD plays a crucial role in cell fate determination during ACD, and the process is regulated by the evolutionarily conserved polarity regulator Par-3 (also known as Pard3) and the dynein motor complex. To visualize the in vivo trafficking patterns of Notch signaling endosomes in mitotic RGPs, we have developed this antibody uptake assay. Using the assay, we have uncovered the dynamicity of DeltaD-containing endosomes during RGP division.

This work develops an antibody uptake assay for imaging intra-lineage Notch/DeltaD signaling in dividing radial glia progenitors of the embryonic zebrafish brain.

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing ...