Name: culture of adult transgenic zebrafish retinal explants for live-cell imaging by multiphoton microscopy
Uploaded: 2017-02-24T15:00:00
Description: Watch this Scientific Journal Video about Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy at JoVE.com

We appreciate the support provided by William Archer and the Notre Dame Integrated Imaging Facility. Special thanks are directed to the Freimann Life Sciences technicians for their continuous help and their care and husbandry of the zebrafish. This study was supported by grants from the National Eye Institute of NIH to DRH (R01-EY018417, R01-EY024519) and the Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN.

Note: Zebrafish were raised and maintained in the Notre Dame Zebrafish facility in the Freimann Life Sciences Center. The methods described in this manuscript are approved by the University of Notre Dame Animal Care and Use Committee and are in compliance with the statement for the use of animals in vision research by the Association for Research in Vision and Ophthalmology.
1. Solutions
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	<li>Prepare 70% ethanol to sterilize the tissue culture hood and any equipment/reagents that are tr...

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	<li>Fausett, B. V., Goldman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+alpha1+tubulin-expressing+Muller+glia+in+regeneration+of+the+injured+zebrafish+retina.">A role for alpha1 tubulin-expressing Muller glia in regeneration of the injured zebrafish retina.</a> J.Neurosci. 26 (23), 6303-6313 (2006).</li><li>Kassen, S. 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=Time+course+analysis+of+gene+expression+during+light-induced+photoreceptor+cell+death+and+regeneration+in+albino+zebrafish.">Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish.</a> Dev.Neurobiol. 67 (8), 1009-1031 (2007).</li><li>Vihtelic, T. S., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+rod+and+cone+cell+death+and+regeneration+in+the+adult+albino+zebrafish+(Danio+rerio)+retina.">Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina.</a> J.Neurobiol. 44 (3), 289-307 (2000).</li><li>Bernardos, R. L., Barthel, L. K., Meyers, J. R., Raymond, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late-stage+neuronal+progenitors+in+the+retina+are+radial+Muller+glia+that+function+as+retinal+stem+cells.">Late-stage neuronal progenitors in the retina are radial Muller glia that function as retinal stem cells.</a> J.Neurosci. 27 (26), 7028-7040 (2007).</li><li>Nelson, C. M., Ackerman, K. M., O'Hayer, P., Bailey, T. J., Gorsuch, R. A., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor-alpha+is+produced+by+dying+retinal+neurons+and+is+required+for+Muller+glia+proliferation+during+zebrafish+retinal+regeneration.">Tumor necrosis factor-alpha is produced by dying retinal neurons and is required for Muller glia proliferation during zebrafish retinal regeneration.</a> J.Neurosci. 33 (15), 6524-6539 (2013).</li><li>Lahne, M., Li, J., Marton, R. M., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin-Cytoskeleton-+and+Rock-Mediated+INM+Are+Required+for+Photoreceptor+Regeneration+in+the+Adult+Zebrafish+Retina.">Actin-Cytoskeleton- and Rock-Mediated INM Are Required for Photoreceptor Regeneration in the Adult Zebrafish Retina.</a> J.Neurosci. 35 (47), 15612-15634 (2015).</li><li>Nagashima, M., Barthel, L. K., Raymond, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+self-renewing+division+of+zebrafish+Muller+glial+cells+generates+neuronal+progenitors+that+require+N-cadherin+to+regenerate+retinal+neurons.">A self-renewing division of zebrafish Muller glial cells generates neuronal progenitors that require N-cadherin to regenerate retinal neurons.</a> Development. 140 (22), 4510-4521 (2013).</li><li>Sauer, M. E., Walker, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioautographic+study+of+interkinetic+nuclear+migration+in+the+neural+tube.">Radioautographic study of interkinetic nuclear migration in the neural tube.</a> Proc.Soc.Exp.Biol.Med. 101 (3), 557-560 (1959).</li><li>Pearson, R. A., Luneborg, N. L., Becker, D. L., Mobbs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions+modulate+interkinetic+nuclear+movement+in+retinal+progenitor+cells.">Gap junctions modulate interkinetic nuclear movement in retinal progenitor cells.</a> J.Neurosci. 25 (46), 10803-10814 (2005).</li><li>Baye, L. M., Link, 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=Interkinetic+nuclear+migration+and+the+selection+of+neurogenic+cell+divisions+during+vertebrate+retinogenesis.">Interkinetic nuclear migration and the selection of neurogenic cell divisions during vertebrate retinogenesis.</a> J.Neurosci. 27 (38), 10143-10152 (2007).</li><li>Del Bene, F., Wehman, A. M., Link, B. 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L., 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=Multiphoton+imaging+of+chick+retinal+development+in+relation+to+gap+junctional+communication.">Multiphoton imaging of chick retinal development in relation to gap junctional communication.</a> J.Physiol. 585 (Pt 3), 711-719 (2007).</li><li>Surzenko, N., Crowl, T., Bachleda, A., Langer, L., Pevny, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX2+maintains+the+quiescent+progenitor+cell+state+of+postnatal+retinal+Muller+glia.">SOX2 maintains the quiescent progenitor cell state of postnatal retinal Muller glia.</a> Development. 140 (7), 1445-1456 (2013).</li><li>Nickerson, P. 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=Live+imaging+and+analysis+of+postnatal+mouse+retinal+development.">Live imaging and analysis of postnatal mouse retinal development.</a> BMC Dev.Biol. 13, 24 (2013).</li><li>Suzuki, S. C., Bleckert, A., Williams, P. R., Takechi, M., Kawamura, S., Wong, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+photoreceptor+types+in+zebrafish+are+generated+by+symmetric+terminal+divisions+of+dedicated+precursors.">Cone photoreceptor types in zebrafish are generated by symmetric terminal divisions of dedicated precursors.</a> Proc.Natl.Acad.Sci.U.S.A. 110 (37), 15109-15114 (2013).</li><li>Johnson, T. V., Martin, K. 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Studies investigating the mechanisms governing regeneration of the damaged adult zebrafish retina predominantly used immunocytochemical methods5,25,26,27,28,29,30. Establishing conditions to culture retinal explants and to perform live-cell imaging on phenomena, such as INM, provide us a tec...

Unlike humans, zebrafish (Danio rerio) exhibit a robust regeneration response upon cell death of retinal neurons1,2,3,4. Tumor necrosis factor &#945;, a signaling molecule that is released from dying retinal neurons induces M&#252;ller glia residing in the basal Inner Nuclear Layer (INL) of the retina, to proliferate5 and produce neuronal progenitor cells that continue to proliferate before differentiating into the neuronal cell types that died2,3,4. During the proliferative phase of the regeneration response, the nuclei of M&#252;ller glia and their derived neuronal progenitor cells undergo a repetitive migratory pattern in phase with the cell cycle (Interkinetic Nuclear Migration, INM)6,7. Nuclei positioned in the basal INL replicate their DNA before migrating to the Outer Nuclear Layer (ONL) where they divide before the arising nuclei return basally to the INL. This process was first described during neuroepithelial development using histological methods, while live-cell imaging approaches later confirmed the interpretation by Sauer8,9,10,11,12. Both histochemical and live-cell imaging approaches have been used to determine mechanisms underlying INM and its function in developing neuroepithelia including the retina9,11,12,13. However, the mechanisms governing INM in the adult regenerating retina have not been studied in much detail6,7. Live-cell imaging will be an invaluable approach to advance our knowledge of the signaling pathways that control INM in the adult regenerating retina.
Until recently, live-cell imaging of INM in the retina was limited to either live zebrafish embryos or to embryonic chick or postnatal mouse retinal explants9,10,11,12,14,15,16. While retinal explants from adult animals of a variety of species including mouse, rat and zebrafish have been utilized for different cell biological approaches17,18,19,20, live-cell imaging experiments using retinal explants have been restricted to brief periods of time and have not been executed continuously over several hours21,22. Here, we describe a detailed protocol to culture light-damaged adult zebrafish retinas to perform live-cell imaging experiments monitoring INM using multi-photon microscopy6. Live-cell imaging approaches are advantageous over immunohistochemical methods when investigating the mechanisms controlling INM, as the dynamics of INM, e.g., velocities might be affected rather than the location of mitosis, which would potentially not be detected using immunocytochemistry.
In the future, this method has also the potential to be modified to study other dynamic processes during retinal regeneration, such as phagocytosis of dying photoreceptors by M&#252;ller glia or the behavior of microglia.

<table border="0" cellpadding="0" cellspacing="0" width="876">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:491px;">Dumont forceps size #5</td>
			<td style="width:201px;">World precision instruments</td>
			<td align="right" style="width:113px;">14098</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont forceps size #5, 45&#176; angle</td>
			<td>World precision instruments</td>
			<td align="right">14101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">McPherson-Vannas scissors</td>
			<td>World precision instruments</td>
			<td align="right">501233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluordishes</td>
			<td>World precision instruments</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereomicroscope</td>
			<td>Nikon&#160;</td>
			<td>SMZ-1B</td>
			<td>similar type of dissection stereomicroscope will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biological Safety Cabinet class type A2</td>
			<td>Labconco</td>
			<td></td>
			<td>equivalent type will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tissue culture incubator</td>
			<td>Thermoscientific</td>
			<td>HEPA-class 100</td>
			<td>equivalent type will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylvania fluorescent lamps OSFP5835HOECO</td>
			<td>Bulbtronics</td>
			<td align="right">31850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 &#181;m pore-size Acrodisc syringe filter</td>
			<td>VWR</td>
			<td align="right">4192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 ml Luer-lok syringe</td>
			<td>VWR</td>
			<td>BD309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 ml Luer-lok syringe</td>
			<td>VWR</td>
			<td>BD309653</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td>FischerScientific</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2</td>
			<td>ThermoScientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgCl2</td>
			<td>EMD Millipore</td>
			<td align="right">5980</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">HBSS w/o Ca2+/Mg2+, w/o phenol red, Gibco</td>
			<td>ThermoScientific</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM w/o phenol red, Gibco</td>
			<td>ThermoScientific</td>
			<td>5100-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horse serum, heat-inactivated</td>
			<td>ThermoScientific</td>
			<td>26050-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">penicillin/streptomycin</td>
			<td>VWR</td>
			<td>16777-164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrapure low melting point agarose</td>
			<td>ThermoScientific</td>
			<td>16520-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol, absolute</td>
			<td>ThermoScientific</td>
			<td>BP2818-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-phenoxyethanol</td>
			<td>Sigma</td>
			<td align="right">77699</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning Cell-Tak cell and tissue adhesive&#160;</td>
			<td>VWR</td>
			<td align="right">354240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">refractive index liquid&#160;</td>
			<td>Cargille Lab</td>
			<td>1803Y</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon A1 multiphoton microscope equipped with a MaiTai infrared laser</td>
			<td>Nikon</td>
			<td></td>
			<td>equivalent system will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40x Apo long-distance water immersion objective (N.A. 1.15)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">environmental chamber equipped with insert for 35 mm petridishes</td>
			<td>Okolab</td>
			<td></td>
			<td>equivalent system will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NIS analysis software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

culture of adult transgenic zebrafish retinal explants for live-cell imaging by multiphoton microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

The isolation of the retina according to the procedure outlined in the schematic in Figure 1 allows the culturing of a flattened dorsal retina from light-damaged adult Tg[gfap:nGFP]mi2004 zebrafish over a period of at least 24 h in a 5% CO2/air environment. These flat-mounted retinal explants can be used to image focal planes at deep tissue levels. An example is M&#252;ller glia/neuronal progenitor cell nuclei labeled with GFP from the M&#25...

Watch this Scientific Journal Video about Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy at JoVE.com

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

The overall goal of this isolation procedure is to enable live cell imaging of the proliferating cells that are undergoing interkinetic nuclear migration in the retinal explant cultures from damaged zebrafish. This method can answer key questions in retinal regeneration, such as what mechanisms drive interkinetic nuclear migration or whether Muller glia in a renal progenitor cell interkinetic nuclear migration are similarly regulated. The main advantage of this technique is that the dynamic behavior of the proliferating cells undergoing interkinetic nuclear migration during regeneration can be monitored, which permits the investigation of cell cycle stage specific mechanisms.Though this method can provide insight into interkinetic nuclear migration, it can also be applied to other retinal research areas, such as investigating microglial behavior or Muller glia phagocytosis of dying neurons. Begin this procedure by transferring the zebrafish onto a dry paper towel. Then, remove one of the eyes with a pair of curved forceps and transfer it onto a FluoroDish.Under a stereo microscope, orient the eye with the pupil facing the coverslip of the FluoroDish so that the optic nerve on the back of the eye is visible. Then, remove the optic nerve as close to the back of the eye as possible. In addition, remove the connective tissue outside of the eye.Hold the eye between its nasal and temporal sides with a pair of forceps while making an incision at the optic stock by piercing through the lamina cribrosa with one scissor blade and cutting along both the nasal and temporal sides of the eye. Next, pull the dorsal and the ventral sides of the retina apart. Remove the sclera from the dorsal retina with one pair of forceps while holding the lens that is connected to the retina with a second pair of forceps.Following that, remove the lens and the vitreous without damaging the retina. Then, flatten the retina with the ganglion cell layer facing the coverslip of the FluoroDish. Afterward, surround the retina with 10 microliters of 1%low melting point agarose.Make sure that the retina is not lifted by the liquid agarose because even slight elevation might affect the ability to focus through the tissue. If lifting is observed, use a pipette to remove the agarose. It is critical that the agarose does not lift the tissue from the coverslip, as this reduces the ability to obtain quality images.Repeat this procedure several times before adding 1%low melting point agarose to cover the entire FluoroDish. Once the agarose has solidified, add 1.5 milliliters of culture medium to the tissue. In the image acquisition software, open A1 MP GUI TI pad, A1 compact GUI, and ND acquisition windows.For multi photon imaging, ensure that the IRNDD option is chosen in A1 compact GUI window. Then, in the setting field, select IRDM for the first dichroic mirror and check that the band pass filter is set to 525 to 50 to acquire GFP fluorescence. Next, switch on the IR laser in the A1 MP GUI window.It will take a few minutes for the laser to be ready. After that, set the wavelength to 910 nanometers to excite GFP flourescence and align the laser by clicking the auto-alignment button. After placing the retinal culture on the multiphoton microscope stage, ensure that the room and equipment lights are switched off before opening the shutter to avoid overexposure of the photo multiplier tube.To reduce noise levels, house the microscope in a darkened environment. Acquire images of a field of view of 300 by 300 pixels at a zoom of two, which are set in the scan area window and a pixel dwelling time of 4.8 microseconds per pixel chosen in the A1 compact GUI window. Roughly set up the laser power by changing the acquisition area in the A1 MP GUI window and the gain in the A1 compact GUI window.Now, focus on the ganglion cell layer and set the top focal plane of the z stack in the z sub-window within the ND acquisition window. Move the focal plane through the level of the outer nuclear layer, which is characterized by the presence of dimly labeled GFAP and GFP positive cells that are round and enlarged relative to their counterparts in the inner nuclear layer. Set this plane as the bottom of the z stack.Next, set the z step size between 0.7 to one micrometer. To set up the z intensity correction, open the z intensity correction window and choose from ND for setting the z stack range. Then, click on the bottom focal plane in the z intensity correction window and set the laser intensity and gain.Afterward, click the arrow next to the z values in the z intensity correction window to confirm the settings that are subsequently shown under device settings in the z intensity correction window for the chosen focal plane. Repeat the process for the middle and top planes, increasing the laser power and gain. Set the acquisition area in the A1 MP GUI window and avoid selecting an acquisition area larger than 15 and a gain higher than 126 at the start of imaging to circumvent photo bleaching and increased noise levels.Subsequently, choose relative intensity correction in the z intensity correction window. In the time series sub window of the ND acquisition window, set the duration to 8 hours and the interval to no delay. Then, click the run z correction in the z stack sub window of the ND acquisition window to acquire 3D time series.In this procedure, crop a region that contains a dividing GFAP and GFP positive nucleus. Choose a cropped region that contains at least one nucleus that does not undergo INM in order to set a reference point to subsequently measure the distances that the dividing nucleus migrated in relation to the basal INL. It helps to prepare a 3D reconstruction.Using the show slices view function, generate orthogonal projections. Subsequently, change the mode from slice to maximum intensity projection. Then, turn off the XY view of the orthogonal maximum projection.Depending on the orientation at which the migrating and dividing nucleus is best visible, also turn off either the XZ or the YZ view. Click on the remaining image with the right mouse button and extract the XZ or YZ image series with the create new document from this view function. If necessary, rotate the image.Using the manual measurement function, draw a horizontal line across the image at the bottom level of the nucleus that remains in the basal INL and does not undergo INM. Measure the distance between the reference line and the basal point of the migrating nucleus for the time series using the line measurement tool in the analysis software. Shown here is the maximum YZ projection of a z stack image series of the retinal explants at different time points of the time lapse recording acquired by multiphoton microscopy.Using the line tool under the manual measurement function, a horizontal line was placed at the level of the reference cell indicated by a star. A vertical measurement line was positioned starting at the horizontal line and extending to the basal position of the migrating GF AP and GFP positive nucleus. Distance that the cell, F0, measured in the previous figure migrated apically and that the arising daughter cells, D1 and D2, migrated basally in the multi photon time lapse recording.The red line indicates the measurements for which the apical migration velocity was calculated, while the black and gray lines indicate the measurements used to calculate the basal migration velocities for D1 and D2 respectively. The distance was plotted against time in itself for apical migration before the nuclear envelope breakdown and the phase of rapid basal migration for daughter cell D1.Once mastered, the isolation of retinas from three fish can be done in 60 to 90 minutes if it is performed properly. This time excludes the preparation steps.While attempting this procedure, it's important to remember to work as quickly as possible to ensure viability of the retinal explant. It is also critical to mount retinas as flat as possible to enable imaging in deeper tissue layers. After watching this video, you should have a good understanding of how to isolate and mount zebrafish retinas, perform live cell imaging in order to monitor interkinetic nuclear migration and to determine migration velocities.

culture-adult-transgenic-zebrafish-retinal-explants-for-live-cell

Research

JoVE Journal

Developmental Biology

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high.
This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

This protocol describes a method to dissect, experimentally manipulate and culture whole retinal explants from chicken embryos. The explant cultures are useful when high success rate, efficacy and reproducibility are needed to test the effects of plasmids for electroporation and/or reagent substances, i.e., enzymatic inhibitors.

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

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 ...